Anti-b7-h3 antibodies and antibody drug conjugates

ABSTRACT

The invention relates to B7 homology 3 protein (B7-H3) antibodies and antibody drug conjugates (ADCs), including compositions and methods of using said antibodies and ADCs.

RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 15/616,901, filed on Jun. 7, 2017, and claims priority to U.S.Provisional Application No. 62/347,476, filed on Jun. 8, 2016, and toU.S. Provisional Application No. 62/366,511, filed on Jul. 25, 2016. Theentire contents of the foregoing applications are expressly incorporatedherein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 7, 2017, isnamed 117813-12603_ST25.txt and is 159,744 bytes in size.

BACKGROUND OF THE INVENTION

The B7 homology 3 protein (B7-H3) (also known as CD276 and B7RP-2, andreferred to herein as “B7-H3”) is a type I transmembrane glycoprotein ofthe immunoglobulin superfamily. Human B7-H3 contains a putative signalpeptide, V-like and C-like Ig domains, a transmembrane region and acytoplasmic domain. Exon duplication in humans results in the expressionof two B7-H3 isoforms having either a single IgV-IgC-like domain(2IgB7-H3 isoform) or a IgV-IgC-IgV-IgC-like domain (4IgB7-H3 isoform)containing several conserved cysteine residues. The predominant B7-H3isoform in human tissues and cell lines is the 4IgB7-H3 isoform.(Steinberger et al., J. Immunol. 172(4): 2352-9 (2004)).

B7-H3 has been reported as having both co-stimulatory and co-inhibitorysignaling functions (see, e.g., Chapoval et al., Nat. Immunol. 2: 269-74(2001); Suh et al., Nat. Immunol. 4: 899-906 (2003); Prasad et al., J.Immunol. 173: 2500-6 (2004); and Wang et al., Eur. J. Immunol. 35:428-38 (2005)). For example, in vitro studies have shown B7-H3'sco-stimulatory function since B7-H3 was able to increase proliferationof cytotoxic T-lymphocytes (CTLs) and upregulate interferon gamma(IFN-γ) production in the presence of anti-CD3 antibody to mimic the Tcell receptor signal (Chapoval et al., 2001). Moreover, in vivo studiesusing cardiac allografts in B7-H3−/− mice showed decreased production ofkey cytokine, chemokine and chemokine receptor mRNA transcripts (e.g.,IL-2, IFN-γ, monocyte chemoattractant protein (MCP-1) and IFN-inducibleprotein (IP)-10) as compared to wild-type control (Wang et al., 2005).In contrast, B7-H3 co-inhibitory function has been observed, forexample, in mice where B7-H3 protein inhibited T-cell activation andeffector cytokine production (Suh et al., 2003). Although no ligandshave been identified for human B7-H3, murine B7-H3 has been found tobind to the triggering receptor expressed on myeloid cells (TREM-) liketranscript 2 (TLT-2), a modulator of adaptive an innate immunitycellular responses. Binding of murine B7-H3 to TLT-2 on CD8⁺ T-cellsenhances T-cell effector functions such as proliferation, cytotoxicityand cytokine production (Hashiguchi et al., Proc. Nat'l. Acad. Sci.U.S.A. 105(30): 10495-500 (2008)).

B7-H3 is not constitutively expressed in many immune cells (e.g.,natural killer (NK) cells, T-cells, and antigen-presenting cells(APCs)), however, its expression can be induced. Further, the expressionof B7-H3 is not restricted to immune cells. B7-H3 transcripts areexpressed in a variety of human tissues including colon, heart, liver,placenta, prostate, small intestine, testis, and uterus, as well asosteoblasts, fibroblasts, epithelial cells, and other cells ofnon-lymphoid lineage, potentially indicating immunological andnon-immunological functions (Nygren et al. Front Biosci. 3:989-93(2011)). However, protein expression in normal tissue is typicallymaintained at a low level and thus, may be subject topost-transcriptional regulation.

B7-H3 is also expressed in a variety of human cancers, including,prostate cancer, clear cell renal cell carcinoma, glioma, melanoma, lungcancer, non-small cell lung cancer (NSCLC), small cell lung cancer,pancreatic cancer, gastric cancer, acute myeloid leukemia (AML),non-Hodgkin's lymphoma (NHL), ovarian cancer, colorectal cancer, coloncancer, renal cancer, hepatocellular carcinoma, kidney cancer, head andneck cancer, hypopharyngeal squamous cell carcinoma, glioblastoma,neuroblastoma, breast cancer, endometrial cancer, and urothelial cellcarcinoma. Although the role of B7-H3 in cancer cells is unclear, itsexpression may orchestrate signaling events that may protect cancercells from innate and adaptive immune responses. For example, B7-H3 isoverexpressed in high-grade prostatic intraepithelial neoplasia andadenocarcinomas of the prostate, and high expression levels of B7-H3 inthese cancerous cells is associated with an increased risk of cancerprogression after surgery (Roth et al. Cancer Res. 67(16): 7893-900(2007)). Further, tumor B7-H3 expression in NSCLC inversely correlatedwith the number of tumor-infiltrating lymphocytes and significantlycorrelated with lymph node metastasis (Sun et al. Lung Cancer 53(2):143-51 (2006)). The level of circulating soluble B7-H3 in NSCLC patientshas also been associated with higher tumor stage, tumor size, lymph nodemetastasis, and distant metastasis (Yamato et al., Br. J. Cancer101(10):1709-16 (2009)).

B7-H3 may also play an important role in T-cell-mediated antitumorresponses in a context dependent manner. For example, gastric cancertumor cell expression of B7-H3 positively correlated with survival time,infiltration depth, and tissue type (Wu et al., World J. Gastroenterol.12(3): 457-9 (2006)). Further, high expression of B7-H3 in pancreatictumor cells was associated with patient survival after surgicalresection and significantly correlated with the number oftumor-infiltrating CD8⁺ T-cells (Loos et al., BMC Cancer 9:463 (2009).

Antibody drug conjugates (ADC) represent a relatively new class oftherapeutics comprising an antibody conjugated to a cytotoxic drug via achemical linker. The therapeutic concept of ADCs is to combine bindingcapabilities of an antibody with a drug, where the antibody is used todeliver the drug to a tumor cell by means of binding to a target surfaceantigen, including target surface antigens that are overexpressed in thetumor cells.

There remains a need in the art for anti-B7-H3 antibodies and anti-B7-H3ADCs that can be used for therapeutic purposes in the treatment ofcancer.

SUMMARY OF THE INVENTION

In certain aspects, the present invention provides for antibodies andantibody drug conjugates (ADCs) that specifically bind to human B7-H3.In certain aspects, the present invention provides novel ADCs that canselectively deliver Bcl-xL inhibitors to target cancer cells, e.g.,B7-H3 expressing cells.

In one aspect, the present invention provides an antoi-B7-H3 antibody,or antigen binding portion thereof, that binds to human B7-H3 (hB7-H3),wherein the antibody, or antigen binding portion thereof, comprises aheavy chain variable region comprising a CDR3 having the amino acidsequence of SEQ ID NO: 12 and a light chain variable region comprising aCDR3 having the amino acid sequence of SEQ ID NO: 15.

In one embodiment, the anti-B7-H3 antibody, or antigen binding portionthereof, comprises a heavy chain variable region comprising a CDR2having the amino acid sequence of SEQ ID NO: 140 and a light chainvariable region comprising a CDR2 having the amino acid sequence of SEQID NO: 7.

In one embodiment, the anti-B7-H3 antibody, or antigen binding portionthereof, comprises a heavy chain variable region comprising a CDR1having the amino acid sequence of SEQ ID NO: 10 and a light chainvariable region comprising a CDR1 having the amino acid sequence ofeither SEQ ID NO: 136 or 138.

In one aspect, the present invention provides an anti-B7-H3 antibody, orantigen binding portion thereof, that binds to human B7-H3 (hB7-H3),wherein the antibody, or antigen binding portion thereof, comprises aheavy chain variable region comprising a CDR3 having the amino acidsequence of SEQ ID NO: 35 and a light chain variable region comprising aCDR3 having the amino acid sequence of SEQ ID NO: 39.

In one embodiment, the anti-B7-H3 antibody, or antigen binding portionthereof, comprises a heavy chain variable region comprising a CDR2having the amino acid sequence of SEQ ID NO: 34, and a light chainvariable region comprising a CDR2 having the amino acid sequence of SEQID NO: 38.

In one embodiment, the anti-B7-H3 antibody, or antigen binding portionthereof, comprises a heavy chain variable region comprising a CDR1having the amino acid sequence of SEQ ID NO: 33 and a light chainvariable region comprising a CDR1 having the amino acid sequence ofeither SEQ ID NO: 37.

In one embodiment, the anti-B7-H3 antibody, or antigen binding portionthereof, is an IgG isotype.

In one embodiment, the anti-B7-H3 antibody, or antigen binding portionthereof, is an IgG1 or an IgG4 isotype.

In one embodiment, the anti-B7-H3 antibody, or antigen binding portionthereof, has a K_(D) of 1.5×10⁻⁸ or less as determined by surfaceplasmon resonance.

In one aspect, the present invention provides an anti-B7-H3 antibody, orantigen-binding portion thereof, that binds to hB7-H3, said antibody, orantigen-binding portion thereof, comprising either (i) a heavy chainvariable region comprising a CDR set of SEQ ID NOs: 10, 11, and 12, anda light chain variable region comprising a CDR set of SEQ ID NOs: 14, 7,and 15, or (ii) a heavy chain variable region comprising a CDR set ofSEQ ID NOs: 33, 34, and 35, and a light chain variable region comprisinga CDR set of SEQ ID NOs: 37, 38, and 39.

In one aspect, the present invention provides an anti-B7-H3 antibodythat binds to human B7-H3 (hB7-H3), wherein the antibody comprises aheavy chain variable region comprising a CDR3 having the amino acidsequence of SEQ ID NO: 12 and a light chain variable region comprising aCDR3 having the amino acid sequence of SEQ ID NO: 15.

In one embodiment, the anti-B7-H3 antibody comprises a heavy chainvariable region comprising a CDR2 having the amino acid sequence of SEQID NO: 140 and a light chain variable region comprising a CDR2 havingthe amino acid sequence of SEQ ID NO: 7.

In one embodiment, the anti-B7-H3 antibody comprises a heavy chainvariable region comprising a CDR1 having the amino acid sequence of SEQID NO: 10 and a light chain variable region comprising a CDR1 having theamino acid sequence of either SEQ ID NO: 136 or 138.

In one aspect, the present invention provides an anti-B7-H3 antibodythat binds to human B7-H3 (hB7-H3), wherein the antibody, or antigenbinding portion thereof, comprises a heavy chain variable regioncomprising a CDR3 having the amino acid sequence of SEQ ID NO: 35 and alight chain variable region comprising a CDR3 having the amino acidsequence of SEQ ID NO: 39.

In one embodiment, the anti-B7-H3 antibody comprises a heavy chainvariable region comprising a CDR2 having the amino acid sequence of SEQID NO: 34, and a light chain variable region comprising a CDR2 havingthe amino acid sequence of SEQ ID NO: 38.

In one embodiment, the anti-B7-H3 antibody comprises a heavy chainvariable region comprising a CDR1 having the amino acid sequence of SEQID NO: 33 and a light chain variable region comprising a CDR1 having theamino acid sequence of either SEQ ID NO: 37.

In one embodiment, the anti-B7-H3 antibody is an IgG isotype.

In one embodiment, the anti-B7-H3 antibody is an IgG1 or an IgG4isotype.

In one embodiment, the anti-B7-H3 antibody has a K_(D) of 1.5×10⁻⁸ orless as determined by surface plasmon resonance.

In one aspect, the present invention provides an anti-B7-H3 antibodythat binds to hB7-H3, said antibody comprising either (i) a heavy chainvariable region comprising a CDR set of SEQ ID NOs: 10, 11, and 12, anda light chain variable region comprising a CDR set of SEQ ID NOs: 14, 7,and 15, or (ii) a heavy chain variable region comprising a CDR set ofSEQ ID NOs: 33, 34, and 35, and a light chain variable region comprisinga CDR set of SEQ ID NOs: 37, 38, and 39.

In one embodiment, the anti-B7-H3 antibody, or antigen binding portionthereof, is humanized.

In one embodiment, the anti-B7-H3 antibody, or antigen binding portionthereof, further comprises a human acceptor framework. In oneembodiment, the human acceptor framework comprises an amino acidsequence selected from the group consisting of SEQ ID Nos: 155,156, 164,165, 166, and 167. In one embodiment, the human acceptor frameworkcomprises at least one framework region amino acid substitution. In oneembodiment, the amino acid sequence of the framework is at least 65%identical to the sequence of said human acceptor framework and comprisesat least 70 amino acid residues identical to said human acceptorframework.

In one embodiment, the human acceptor framework comprises at least oneframework region amino acid substitution at a key residue, said keyresidue selected from the group consisting of a residue adjacent to aCDR; a glycosylation site residue; a rare residue; a residue capable ofinteracting with human B7-H3; a residue capable of interacting with aCDR; a canonical residue; a contact residue between heavy chain variableregion and light chain variable region; a residue within a Vernier zone;and a residue in a region that overlaps between a Chothia-definedvariable heavy chain CDR1 and a Kabat-defined first heavy chainframework. In one embodiment, the key residue is selected from the groupconsisting of 48H, 67H, 69H, 71H, 73H, 94H, and 2L. In one embodiment,the key residue substitution is in the variable heavy chain region andis selected from the group consisting of M48I, V67A, I69L, A71V, K73R,and R94G. In one embodiment, the key residue substitution is in thevariable light chain region and is I2V.

In one aspect, the present invention provides an anti-B7-H3 antibody, orantigen-binding portion thereof, that binds to hB7-H3 comprising a heavychain variable region comprising a CDR set of SEQ ID NOs: 25, 26, and27, and a light chain variable region comprising a CDR set of SEQ IDNOs: 29, 30, and 31. In one embodiment, the anti-B7-H3 antibody, orantigen binding portion thereof, is humanized. In one embodiment, theanti-B7-H3 antibody, or antigen binding portion thereof, furthercomprises a human acceptor framework.

In one embodiment, the human acceptor framework comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 155 to 158.In one embodiment, the human acceptor framework comprises at least oneframework region amino acid substitution. In one embodiment, the aminoacid sequence of the framework is at least 65% identical to the sequenceof said human acceptor framework and comprises at least 70 amino acidresidues identical to said human acceptor framework. In one embodiment,the amino acid sequence of the framework is at least 85% identical. 90%identical, 95% identical, 96% identical, 97% identical, 98% identical,or 99% identical to the sequence of the human acceptor framework andcomprises at least 70, at least 75, at least 80, or at least 85 aminoacid residues identical to the human acceptor framework.

In one embodiment, the human acceptor framework comprises at least oneframework region amino acid substitution at a key residue, said keyresidue selected from the group consisting of: a residue adjacent to aCDR; a glycosylation site residue; a rare residue; a residue capable ofinteracting with human B7-H3; a residue capable of interacting with aCDR; a canonical residue; a contact residue between heavy chain variableregion and light chain variable region; a residue within a Vernier zone;and a residue in a region that overlaps between a Chothia-definedvariable heavy chain CDR1 and a Kabat-defined first heavy chainframework. In one embodiment, the key residue is selected from the groupconsisting of 69H, 46L, 47L, 64L, and 71L. In one embodiment, the keyresidue substitution is in the variable heavy chain region and is L69I.In one embodiment, the key residue substitution is in the variable lightchain region and is selected from the group consisting of L46P, L47W,G64V, and F71H.

In one aspect, the present invention provides an anti-hB7-H3 antibody,or antigen-binding portion thereof, comprising a heavy chain CDR1comprising an amino acid sequence as set forth in SEQ ID NO: 10, a heavychain CDR2 comprising an amino acid sequence as set forth in SEQ ID NO:140, a heavy chain CDR3 comprising an amino acid sequence as set forthin SEQ ID NO: 12, a light chain CDR1 comprising an amino acid sequenceas set forth in SEQ ID NO: 136 or 138, a light chain CDR2 comprising anamino acid sequence as set forth in SEQ ID NO: 7, and a light chain CDR3comprising an amino acid sequence as set forth in SEQ ID NO: 15.

In another aspect, the present invention provides an anti-hB7-H3antibody, or antigen-binding portion thereof, comprising a heavy chainCDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 33, aheavy chain CDR2 comprising an amino acid sequence as set forth in SEQID NO: 34, a heavy chain CDR3 comprising an amino acid sequence as setforth in SEQ ID NO: 35, a light chain CDR1 comprising an amino acidsequence as set forth in SEQ ID NO: 37, a light chain CDR2 comprising anamino acid sequence as set forth in SEQ ID NO: 38, and a light chainCDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 39.

In one embodiment, the anti-hB7-H3 antibody, or antigen-binding portionthereof, comprises a heavy chain variable domain comprising an aminoacid sequence set forth in SEQ ID NO: 139 and a light chain variabledomain comprising an amino acid sequence set forth in SEQ ID NO: 135.

In one embodiment, the anti-hB7-H3 antibody, or antigen-binding portionthereof, comprises a heavy chain comprising an amino acid sequencehaving at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:139, and/or a light chain comprising an amino acid sequence having atleast 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 135.

In one embodiment, the anti-hB7-H3 antibody, or antigen-binding portionthereof, comprises a heavy chain variable domain comprising an aminoacid sequence set forth in SEQ ID NO: 139 and a light chain variabledomain comprising an amino acid sequence set forth in SEQ ID NO: 137.

In one embodiment, the anti-hB7-H3 antibody, or antigen-binding portionthereof, comprises a heavy chain comprising an amino acid sequencehaving at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:139, and/or a light chain comprising an amino acid sequence having atleast 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 137.

In one embodiment, the anti-hB7-H3 antibody, or antigen-binding portionthereof, comprises a heavy chain variable domain comprising an aminoacid sequence set forth in SEQ ID NO: 147 and a light chain variabledomain comprising an amino acid sequence set forth in SEQ ID NO: 144.

In one embodiment, the anti-hB7-H3 antibody, or antigen-binding portionthereof, comprises a heavy chain comprising an amino acid sequencehaving at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:147, and/or a light chain comprising an amino acid sequence having atleast 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 144.

In one aspect, the present invention provides an anti-hB7-H3 antibodycomprising a heavy chain CDR1 comprising an amino acid sequence as setforth in SEQ ID NO: 10, a heavy chain CDR2 comprising an amino acidsequence as set forth in SEQ ID NO: 140, a heavy chain CDR3 comprisingan amino acid sequence as set forth in SEQ ID NO: 12, a light chain CDR1comprising an amino acid sequence as set forth in SEQ ID NO: 136 or 138,a light chain CDR2, comprising an amino acid sequence as set forth inSEQ ID NO: 7, and a light chain CDR3 comprising an amino acid sequenceas set forth in SEQ ID NO: 15.

In another aspect, the present invention provides an anti-hB7-H3antibody comprising a heavy chain CDR1 comprising an amino acid sequenceas set forth in SEQ ID NO: 33, a heavy chain CDR2 comprising an aminoacid sequence as set forth in SEQ ID NO: 34, a heavy chain CDR3comprising an amino acid sequence as set forth in SEQ ID NO: 35, a lightchain CDR1 comprising an amino acid sequence as set forth in SEQ ID NO:37, a light chain CDR2, comprising an amino acid sequence as set forthin SEQ ID NO: 38, and a light chain CDR3 comprising an amino acidsequence as set forth in SEQ ID NO: 39.

In one embodiment, the anti-hB7-H3 antibody comprises a heavy chainvariable domain comprising an amino acid sequence set forth in SEQ IDNO: 139 and a light chain variable domain comprising an amino acidsequence set forth in SEQ ID NO: 135.

In one embodiment, the anti-hB7-H3 antibody comprises a heavy chaincomprising an amino acid sequence having at least 90%, 95%, 96%, 97%,98%, or 99% identity to SEQ ID NO: 139, and/or a light chain comprisingan amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 135.

In one embodiment, the anti-hB7-H3 antibody comprises a heavy chainvariable domain comprising an amino acid sequence set forth in SEQ IDNO: 139 and a light chain variable domain comprising an amino acidsequence set forth in SEQ ID NO: 137.

In one embodiment, the anti-hB7-H3 antibody comprises a heavy chaincomprising an amino acid sequence having at least 90%, 95%, 96%, 97%,98%, or 99% identity to SEQ ID NO: 139, and/or a light chain comprisingan amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 137.

In one embodiment, the anti-hB7-H3 antibody comprises a heavy chainvariable domain comprising an amino acid sequence set forth in SEQ IDNO: 147 and a light chain variable domain comprising an amino acidsequence set forth in SEQ ID NO: 144.

In one embodiment, the anti-hB7-H3 antibody comprises a heavy chaincomprising an amino acid sequence having at least 90%, 95%, 96%, 97%,98%, or 99% identity to SEQ ID NO: 147, and/or a light chain comprisingan amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 144.

In one embodiment, the anti-hB7-H3 antibody, or antigen-binding portionthereof, comprises a heavy chain CDR set corresponding to antibodyhuAb13v1, and a light chain CDR set corresponding to antibody huAb13v71.In one embodiment, the anti-hB7-H3 antibody, or antigen-binding portionthereof, comprises a heavy chain variable region corresponding toantibody huAb13v1, and a light chain variable region corresponding toantibody huAb13v1.

In one embodiment, the anti-hB7-H3 antibody, or antigen-binding portionthereof, comprises a heavy chain CDR set corresponding to antibodyhuAb3v2.5, and a light chain CDR set corresponding to antibodyhuAb3v2.5. In one embodiment, the anti-hB7-H3 antibody, orantigen-binding portion thereof, comprises a heavy chain variable regioncorresponding to antibody huAb3v2.5, and a light chain variable regioncorresponding to antibody huAb3v2.5.

In one embodiment, the antibody, or antigen binding portion thereof,hinds cynomolgus B7-H3.

In one embodiment, the antibody, or antigen binding portion thereof, hasa dissociation constant (K_(D)) to hB7-H3 selected from the groupconsisting of: at most about 10⁻¹⁰ M; at most about 14⁻⁸ M; at mostabout 10⁻⁹M; at most about 10⁻¹⁰ M; at most about 10⁻¹¹ M; at most about10⁻¹² M; and at most 10⁻¹³ M.

In one embodiment, the antibody, or antigen binding portion thereof,comprises a heavy chain immunoglobulin constant domain of a human IgMconstant domain, a human IgG1 constant domain, a human IgG2 constantdomain, a human IgG3 constant domain, a human IgG4 constant domain, ahuman IgA constant domain, or a human IgE constant domain.

In one embodiment, the antibody is an IgG having four polypeptide chainswhich are two heavy chains and two light chains.

In one embodiment, the antibody, or antigen-binding portion thereof,comprises the heavy chain immunoglobulin constant region domain is ahuman IgG1 constant domain. In one embodiment, the human IgG1 constantdomain comprises an amino acid sequence of SEQ ID NO: 159 or SEQ ID NO:160.

In one aspect, the present invention provides an isolated antibody, orantigen binding portion thereof, that binds to human B7-H3 (1197-H3),wherein the antibody, or antigen binding portion thereof, comprises aheavy chain comprising the amino acid sequence of SEQ ID NO: 168 and alight chain comprising the amino acid sequence of SEQ ID NO: 169.

In one aspect, the present invention provides an isolated antibody, orantigen binding portion thereof, that binds to human B7-H3 (hB7-H3),wherein the antibody, or antigen binding portion thereof, comprises aheavy chain comprising the amino acid sequence of SEQ ID NO: 170 and alight chain comprising the amino acid sequence of SEQ ID NO: 171.

In one aspect, the present invention provides an isolated antibody, orantigen binding portion thereof, that binds to human B7-H3 (hB7-H3),wherein the antibody, or antigen binding portion thereof, comprises aheavy chain comprising the amino acid sequence of SEQ ID NO: 172 and alight chain comprising the amino acid sequence of SEQ ID NO: 173.

In one embodiment, the antibody, or antigen binding portion thereof,further comprises a light chain immunoglobulin constant domaincomprising a human Ig kappa constant domain or a human Ig lambdaconstant domain.

In one embodiment, the anti-hB7-H3 antibody, or antigen-binding portionthereof, competes with the antibody, or antigen binding portion thereof,of any one of the anti-hB7-H3 antibodies, or antigen-binding portionsthereof, disclosed herein.

In one aspect, the present invention provides a pharmaceuticalcomposition comprising the anti-hB7-H3 antibody, or antigen bindingportion thereof, as disclosed herein, and a pharmaceutically acceptablecarrier.

In another aspect, the present invention provides an anti-hB7-H3Antibody Drug Conjugate (ADC) comprising an anti-hB7-H3 antibodydisclosed herein conjugated to a drug via a linker. In one embodiment,the drug is an auristatin or a pyrrolobenzodiazepine (PBD). In oneembodiment, the drug is a Bcl-xL inhibitor.

In one aspect, the present invention provides an anti-hB7-H3 antibodydrug conjugate (ADC) comprising a drug linked to an anti-human B7-H3(hB7-H3) antibody by way of a linker, wherein the drug is a Bcl-xLinhibitor according to structural formula (IIa), (IIb), (IIc), or (IId):

wherein:

Ar¹ is selected from

and is optionally substituted with one or more substituentsindependently selected from halo, hydroxy, nitro, lower alkyl, lowerheteroalkyl, C₁₋₄alkoxy, amino, cyano and halomethyl;

Ar² is selected from

or an N-oxide thereof, and is optionally substituted with one or moresubstituents independently selected from halo, hydroxy, nitro, loweralkyl, lower heteroalkyl, C₁₋₄alkoxy, amino, cyano and halomethyl,wherein the R¹²—Z^(2b)—, R′—Z^(2b)—, #—N(R⁴)—R¹³—Z^(2b)—, or#—R′—Z^(2b)— substituents are attached to Ar² at any Ar² atom capable ofbeing substituted; Z¹ is selected from N, CH, C-halo, C—CH₃ and C—CN;Z^(2a) and Ar² are each independently from one another, selected from abond, NR⁶, CR^(6a)R^(6b), O, S, S(O), S(O)₂, —NR⁶C(O)—,—NR^(6a)C(O)NR^(6b)—, and NR⁶C(O)O—;

R′ is

wherein #, where attached to R′, is attached to R′ at any R′ atomcapable of being substituted; X′ is selected at each occurrence from—N(R¹⁰)—N(R¹⁰)C(O)—, —N(R¹⁰)S(O)₂—, S(O)₂N(R¹⁰)—, and —O—; n is selectedfrom 0-3; R¹⁰ is independently selected at each occurrence fromhydrogen, lower alkyl, heterocycle, aminoalkyl, G-alkyl, and—(CH₂)₂—O—(CH₂)₂—O—(CH₂)₂—NH₂; G at each occurrence is independentlyselected from a polyol, a polyethylene glycol with between 4 and 30repeating units, a salt and a moiety that is charged at physiologicalpH; SP^(a) is independently selected at each occurrence from oxygen,—S(O)₂N(H)—, —N(H)S(O)₂—, —N(H)C(O)—, —C(O)N(H)—, —N(H)—, arylene,heterocyclene, and optionally substituted methylene; wherein methyleneis optionally substituted with one or more of —NH(CH₂)₂G, NH₂,C₁₋₈alkyl, and carbonyl; m² is selected from 0-12; R¹ is selected fromhydrogen, methyl, halo, halomethyl, ethyl, and cyano; Fe is selectedfrom hydrogen, methyl, halo, halomethyl and cyano; R³ is selected fromhydrogen, methyl, ethyl, halomethyl and haloethyl; R⁴ is selected fromhydrogen, lower alkyl and lower heteroalkyl or is taken together with anatom of R¹³ to form a cycloalkyl or heterocyclyl ring having between 3and 7 ring atoms; R⁶, R^(6a) and R^(6b) are each, independent from oneanother, selected from hydrogen, optionally substituted lower alkyl,optionally substituted lower heteroalkyl, optionally substitutedcycloalkyl and optionally substituted heterocyclyl, or are takentogether with an atom from R⁴ and an atom from R¹³ to form a cycloalkylor heterocyclyl ring having between 3 and 7 ring atoms; R^(11a) andR^(11b) are each, independently of one another, selected from hydrogen,halo, methyl, ethyl, halomethyl, hydroxyl, methoxy, CN, and SCH₃, R¹² isoptionally R′ or is selected from hydrogen, halo, cyano, optionallysubstituted alkyl, optionally substituted heteroalkyl, optionallysubstituted heterocyclyl, and optionally substituted cycloalkyl; R¹³ isselected from optionally substituted C₁₋₈ alkylene, optionallysubstituted heteroalkylene, optionally substituted heterocyclene, andoptionally substituted cycloalkylene; and 4 represents a point ofattachment to a linker; and wherein the anti-hB7-H3 antibody binds toB7-H3 (SEQ ID NO: 149) with a dissociation constant (K_(d)) of about1×10⁻⁶ M or less. In a further embodiment, the antibody comprises aheavy chain variable domain comprising the amino acid sequence set forthin SEQ ID NO: 147 and a light chain variable domain comprising the aminoacid sequence set forth in SEQ ID NO: 144; binds to B7-H3 (SEQ ID NO:149) with a dissociation constant (K_(d)) of about 1×10⁻⁶ M or less, asdetermined by surface plasmon resonance; and/or inhibits tumor growth inan in vivo human small-cell lung carcinoma (SCLC) xenograft assay with atumor growth inhibition % (TGI %) of at least about 50% relative to ahuman IgG antibody which is not specific for B7-H3, wherein the humanIgG antibody is administered in the SCLC xenograft assay at the samedose and frequency as the anti-hB7-H3 antibody.

In one embodiment, the ADC is a compound according to structural formula(I):

(D-L-LK_(m)Ab  (I)

wherein D is the Bcl-xL inhibitor drug of formula (IIa), (IIb), (IIc) or(IId); L is the linker; Ab is the anti-hB7-H3 antibody; LK represents acovalent linkage linking the linker (L) to the anti-hB7-3 antibody (Ab);and m is an integer ranging from 1 to 20.

In one embodiment, G at each occurrence is a salt or a moiety that ischarged at physiological pH.

In one embodiment, G at each occurrence is a salt of a carboxylase, asulfonate, a phosphonate, or ammonium.

In one embodiment, G at each occurrence is a moiety that is charged atphysiological pH selected from the group consisting of carboxylate, asulfonate, a phosphonate, and an amine.

In one embodiment, G at each occurrence is a moiety containing apolyethylene glycol with between 4 and 30 repeating units, or a polyol.

In one embodiment, the polyol is a sugar.

In one embodiment, the ADC is of the formula (IIa) or formula (IId), andR′ includes at least one substitutable nitrogen suitable for attachmentto a linker.

In one embodiment, G is selected at each occurrence from:

wherein M is hydrogen or a positively charged counterion.

In one embodiment. R′ is selected from

wherein # represents either a hydrogen atom in the Bcl-xL inhibitor drugof the ADCs of formula (IIb) or (IIc) or the point of attachment in theBcl-xL inhibitor drug of the ADCs of formula (IIa) or (IId) to a linkerL.

In one embodiment, Ar¹ is selected from

and is optionally substituted with one or more substituentsindependently selected from halo, cyano, methyl, and halomethyl.

In one embodiment. Ar¹ is

In one embodiment. Ar² is

optionally substituted with one or more substituents.

In one embodiment, Ar² is selected from

and is optionally substituted with one or more substituents.

In one embodiment, Ar² is substituted with one or more solubilizinggroups.

In one embodiment, each solubilizing group is, independently of theothers, selected from a moiety containing a polyol, a polyethyleneglycol with between 4 and 30 repeating units, a salt, or a moiety thatis charged at physiological pH.

In one embodiment, Ar² is substituted with one or more solubilizinggroups.

In one embodiment, each solubilizing group is, independently of theothers, selected from a moiety containing a polyol, a polyethyleneglycol with between 4 and 30 repeating units, a salt, or a moiety thatis charged at physiological pH.

In one embodiment. Z¹ is N.

In one embodiment, Z^(2a) is O.

In one embodiment, R¹ is methyl or chloro.

In one embodiment, R² is hydrogen or methyl.

In one embodiment, R² is hydrogen.

In one embodiment, Z^(2b) is O.

In one embodiment, Z^(2b) is NH or CH₂.

In one embodiment, the ADC is a compound according to structural formula(IIa).

In one embodiment, the ADC is a compound according to structural formula(IIa) which includes a core selected from structures (C.1)-(C.21):

In one embodiment, the ADC is a compound according to structural formula(IIa.1):

wherein Y is optionally substituted C₁-C₈ alkylene; r is 0 or 1; and sis 1, 2 or 3.

In one embodiment, the ADC is a compound according to structural formula(IIa.2):

wherein U is selected from N, O and CH, with the proviso that when U isO, then V^(a) and R^(21a) are absent; R²⁰ is selected from H and C₁-C₄alkyl; R^(21a) a and R^(21b) are each, independently from one another,absent or selected from H, C₁-C₄ alkyl and G, where G is selected from apolyol, PEG4-30, a salt and a moiety that is charged at physiologicalpH; V^(a) and V^(b) are each, independently from one another, absent orselected from a bond, and an optionally substituted alkylene; R²⁰ isselected from and C₁-C₄ alkyl; and s is 1, 2 or 3.

In one embodiment, the ADC is a compound according to structural formula(IIa.3):

wherein R^(b) is selected from H, C₁-C₄ alkyl and J^(b)-G or isoptionally taken together with an atom of T to form a ring havingbetween 3 and 7 atoms; J^(a) and J^(b) are each, independently from oneanother, selected from optionally substituted C₁-C₈ alkylene andoptionally substituted phenylene; T is selected from optionallysubstituted C₁-C₈ alkylene, CH₂CH₂OCH₂CH₂OCH₂CH₂,CH₂CH₂OCH₂CH₂OCH₂CH₂OCH₂ and a polyethylene glycol containing from 4 to10 ethylene glycol units; G is selected from a polyol, PEG4-30, a saltand a moiety that is charged at physiological pH; and s is 1, 2 or 3.

In one embodiment, the ADC is a compound according to structural formula(IIb).

In one embodiment, the ADC is a compound according to structural formula(IIb.1):

wherein Y is optionally substituted C₁-C₈ alkylene; G is selected from apolyol, PEG4-30, a salt and a moiety that is charged at physiologicalpH; r is 0 or 1; and s is 1, 2 or 3.

In one embodiment, the ADC is a compound according to structural formula(IIc).

In one embodiment, the ADC is a compound according to stnmctural formula(IIc.1):

wherein Y^(a) is optionally substituted C₁-C₈ alkylene; Y^(b) isoptionally substituted C₁-C₈ alkylene; R²³ is selected from H and C₁-C₄alkyl; and G is selected from a polyol, PEG4-30, a salt and a moietythat is charged at physiological pH.

In one embodiment, the ADC is a compound according to structural formula(IIc.2):

wherein Y^(a) is optionally substituted C₁-C₈ alkylene; Y^(b) isoptionally substituted C₁-C₈ alkylene; Y^(c) is optionally substitutedC₁-C₈ alkylene; R²³ is selected from H and C₁-C₄ alkyl; R²⁵ is Y^(b)-Gor is taken together with an atom of Y^(c) to form a ring having 4-6ring atoms; and G is selected from a polyol, PEG4-30, a salt and amoiety that is charged at physiological pH.

In one embodiment, the Bcl-xL inhibitor is selected from the groupconsisting of the following compounds modified in that the hydrogencorresponding to the # position of structural formula (IIa), (IIb),(IIc), or (IId) is not present forming a monoradical:

-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-[1-({3-[2-({2-[2-(carboxymethoxy)ethoxy]ethyl}amino)ethoxy]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   2-{([(2-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]amino}ethyl)sulfonyl]amino}-2-deoxy-D-glucopyranose;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(4-{[(3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl]methyl}benzyl)amino]ethoxy}tricyclo[3.3.1.13,7]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(3-sulfopropyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(H)-yl]-3-{1-[(3-{2-[(2,3-dihydroxypropyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   2-({[4-({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]amino}methyl)phenyl]sulfonyl}amino)-2-deoxy-beta-D-glucopyranose;-   8-(1,3-benzothiazol-2-ylcarbamoyl)-2-{6-carboxy-5-[1-({3-[2-({2-[1-(beta-D-glucopyranuronosyl)-1H-1,2,3-triazol-4-yl]ethyl}amino)ethoxy]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridin-2-yl}-1,2,3,4-tetrahydroisoquinoline;-   3-[1-({3-[2-(2-{[4-(beta-D-allopyranosyloxy)benzyl]amino}ethoxy)ethoxy]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3,5-dimethyl-7-(2-{2-[(2-sulfoethyl)amino]ethoxy}ethoxy)tricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-phosphonoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[methyl(3-sulfo-L-alanyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(3-phosphonopropyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(3-sulfo-L-alanyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3,5-dimethyl-7-(2-{2-[(3-phosphonopropyl)amino]ethoxy}ethoxy)tricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylic    acid;-   3-{1-[(3-{2-[L-alpha-aspartyl(methyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylic    acid;-   6-{4-[({2-[2-(2-aminoethoxy)ethoxy]ethyl}[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]amino)methyl]benzyl}-2,6-anhydro-L-gulonic    acid;-   4-({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)-]dec-1-yl}oxy)ethyl]amino}methyl)phenyl    hexopyranosiduronic acid;-   6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-phosphonoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]-3-{1-[(3,5-dimethyl-7-{2-[methyl(3-sulfo-L-alanyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-([1,3]thiazolo[5,4-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylic    acid;-   3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-([1,3]thiazolo[4,5-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylic    acid;-   6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[(2-carboxyethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(3-phosphonopropyl)(piperidin-4-yl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   3-{-[(3-{2-[D-alpha-aspartyl(methyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3-(2-{[1-(carboxymethyl)piperidin-4-yl]amino}ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylic    acid;-   N-[(5S)-5-amino-6-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](methyl)amino}-6-oxohexyl]-N,N-dimethylmethanaminium;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[piperidin-4-yl(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methy]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-5-(3-phosphonopropoxy)-3,4-dihydroisoquinolin-2(1H)-yl]-3-[1-({3,5-dimethyl-7-[2-(methylamino)ethoxy]tricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3-(2-{[N-(2-carboxyethyl)-L-alpha-aspartyl]amino}ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylic    acid;-   3-{1-[(3-{2-[(2-aminoethyl)(2-sulfoethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylic    acid;-   6-[5-(2-aminoethoxy)-8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-[1-({3,5-dimethyl-7-[2-(methylamino)ethoxy]tricyclo[3.3.1.1^(3,7)]dec-1-yl})methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-3-{1-[(3,5-dimethyl-7-{2-[(3-sulfopropyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{-[(3-{2-[(2-carboxyethyl)(piperidin-4-yl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(3-sulfo-L-alanyl)(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[{2-[(2-carboxyethyl)amino]ethyl}(2-sulfoethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   3-{1-[(3,5-dimethyl-7-{2-[(3-phosphonopropyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-([1,3]thiazolo[4,5-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylic    acid;-   3-{1-[(3,5-dimethyl-7-{2-[(3-phosphonopropyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-([1,3]thiazolo[5,4-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-5-(carboxymethoxy)-3,4-dihydroisoquinolin-2(1H)-yl]-3-[1-({3,5-dimethyl-7-[2-(methylamino)ethoxy]tricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(H)-yl]-3-{1-[(3-{2-[(3-carboxypropyl)(piperidin-4-yl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   3-{1-[(3-{2-[L-alpha-aspartyl(2-sulfoethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylic    acid-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[(1,3-dihydroxypropan-2-yl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[5-(2-aminoethoxy)-8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{(2-[methyl(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-5-{2-[(2-sulfoethyl)amino]ethoxy}-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[methyl(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methy]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl){2-[(2-sulfoethyl)amino]ethyl}amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-5-{2-[(2-carboxyethyl)amino]ethoxy}-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[methyl(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   3-{(1-[(3,5-dimethyl-7-{2-[(3-phosphonopropyl)(piperidin-4-yl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-([1,3]thiazolo[4,5-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylic    acid;-   6-[4-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydro-2H-1,4-benzoxazin-6-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-5-(3-sulfopropoxy)-3,4-dihydroisoquinolin-2(1H)-yl]-3-[1-({3,5-dimethyl-7-[2-(methylamino)ethoxy]tricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylic    acid;-   3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[1-([1,3]thiazolo[4,5-b]pyridin-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]pyridine-2-carboxylic    acid;-   3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-([1,3]thiazolo[4,5-b]pyridin-2-ylcarbamoyl)naphthalen-2-yl]pyridine-2-carboxylic    acid;-   (1ζ)-1-({2-[5-(1-{[3-(2-aminoethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)-6-carboxypyridin-2-yl]-8-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroisoquinolin-5-yl}methyl)-1,5-anhydro-D-glucitol;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[(3-carboxypropyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-3-{1-[(3,5-dimethyl-7-{2-[(3-phosphonopropyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3-(2-{[4-(beta-D-glucopyranosyloxy)benzyl]amino}ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylic    acid;-   3-(1-{[3-(2-{[4-(beta-D-allopyranosyloxy)benzyl]amino}ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylic    acid;-   3-{1-[(3-{2-[azetidin-3-yl(2-sulfoethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylic    acid;-   3-{1-[(3-{2-[(3-aminopropyl)(2-sulfoethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylic    acid;-   6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]-3-{1-[(3-{2-[(2-carboxyethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[(N⁶,N⁶-dimethyl-L-lysyl)(methyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   3-{1-[(3-{2-[(3-aminopropyl)(methyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]pyridine-2-carboxylic    acid;-   3-{1-[(3-{2-[azetidin-3-yl(methyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]pyridine-2-carboxylic    acid;-   N⁶-(37-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-yl)-L-lysyl-N-[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]-L-alaninamide;-   methyl    6-[4-(3-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]amino}propyl)-1H-1,2,3-triazol-1-yl]-6-deoxy-beta-L-glucopyranoside;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-3-{1-[(3-{2-[(2-carboxyethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[5-(1,3-benzothiazol-2-ylcarbamoyl)quinolin-3-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[4-(1,3-benzothiazol-2-ylcarbamoyl)quinolin-6-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[5-(1,3-benzothiazol-2-ylcarbamoyl)quinolin-3-yl]-3-{1-[(3-{2-[(2-carboxyethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   8-(1,3-benzothiazol-2-ylcarbamoyl)-2-{6-carboxy-5-[1-({3-[2-({3-[1-(beta-D-glucopyranuronosyl)-1H-1,2,3-triazol-4-yl]propyl}amino)ethoxy]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridin-2-yl}-1,2,3,4-tetrahydroisoquinoline;-   6-[7-(1,3-benzothiazol-2-ylcarbamoyl)-1H-indol-2-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-6-[3-(methylamino)propyl]-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methy]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   5-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]amino}-5-deoxy-D-arabinitol;-   1-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]amino}-1,2-dideoxy-D-arabino-hexitol;-   6-[4-(1,3-benzothiazol-2-ylcarbamoyl)isoquinolin-6-yl]-3-{1-[(3,5-dimethyl-7-{(2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3-(2-{[3-hydroxy-2-(hydroxymethyl)propyl]amino}ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylic    acid;-   1-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]amino}-1,2-dideoxy-D-erythro-pentitol;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3,5-dimethyl-7-(2-{[(2S,3S)-2,3,4-trihydroxybutyl]amino}ethoxy)tricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3-(2-{[(2S,3S,4R,5R,6R)-2,3,4,5,6,7-hexahydroxyheptyl]amino}ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[({3-[(1,3-dihydroxypropan-2-yl)amino]propyl)sulfonyl}amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[(3-{[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino}-3-oxopropyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3-(2-{[(3S)-3,4-dihydroxybutyl]amino}ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylic    acid;-   4-({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]amino}methyl)phenyl    beta-D-glucopyranosiduronic acid;-   3-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]amino}propyl    beta-D-glucopyranosiduronic acid;-   6-[4-(1,3-benzothiazol-2-ylcarbamoyl)-2-oxidoisoquinolin-6-yl]-3-[1-({3,5-dimethyl-7-[2-(methylamino)ethoxy]tricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylic    acid;-   6-{8-[(1,3-benzothiazol-2-yl)carbamoyl]-3,4-dihydroisoquinolin-2(1H)-yl}-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]acetamido}tricyclo[3.3.1.1^(3,7)]decan-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3,5-dimethyl-7-({2-[(2-sulfoethyl)amino]ethyl}sulfanyl)tricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylic    acid; and-   6-{8-[(1,3-benzothiazol-2-yl)carbamoyl]-3,4-dihydroisoquinolin-2(1H)-yl}-3-{1-[(3,5-dimethyl-7-{3-[(2-sulfoethyl)amino]propyl}tricyclo[3.3.1.1^(3,7)]decan-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid.

In one embodiment, the linker is cleavable by a lysosomal enzyme. In oneembodiment, the lysosomal enzyme is Cathepsin B.

In one embodiment, the linker comprises a segment according tostructural formula (IVa), (IVb), (IVc), or (IVd):

wherein peptide represents a peptide (illustrated N→C, wherein peptideincludes the amino and carboxy “termini”) a cleavable by a lysosomalenzyme; T represents a polymer comprising one or more ethylene glycolunits or an alkylene chain, or combinations thereof;

R^(a) is selected from hydrogen, C₁₋₆alkyl, SO₃H and CH₂SO₃H; R^(y) ishydrogen or C₁₋₄ alkyl-(O)_(r)—(C₁₋₄ alkylene)_(s)-G¹ or C₁₋₃alkyl-(N)—[(C₁₋₄ alkylene)-G¹]₂; R^(z) is C₁₋₄ alkyl-(O)_(r)—(C₁₋₄alkylene)_(s)-G²; G¹ is SO₃H, CO₂H, PEG 4-32, or sugar moiety; G² isSO₃H, CO₂H, or PEG 4-32 moiety; r is 0 or 1; s is 0 or 1; p is aninteger ranging from 0 to 5; q is 0 or 1; x is 0 or 1; y is 0 or 1;

represents the point of attachment of the linker to the Bcl-xLinhibitor; and * represents the point of attachment to the remainder ofthe linker.

In one embodiment, the peptide is selected from the group consisting ofVal-Cit; Cit-Val; Ala-Ala; Ala-Cit; Cit-Ala; Asn-Cit; Cit-Asn; Cit-Cit;Val-Glu; Glu-Val; Ser-Cit; Cit-Ser; Lys-Cit; Cit-Lys; Asp-Cit; Cit-Asp;Ala-Val; Val-Ala; Phe-Lys; Lys-Phe; Val-Lys; Lys-Val; Ala-Lys; Lys-Ala;Phe-Cit; Cit-Phe; Leu-Cit; Cit-Leu; Cit-Ile; Ile-Cit; Phe-Arg; Arg-Phe;Cit-Trp; and Trp-Cit.

In one embodiment, the lysosomal enzyme is β-glucuronidase orβ-galactosidase.

In one embodiment, the linker comprises a segment according tostructural formula (Va), (Vb), (Vc), (Vd), or (Ve):

wherein q is 0 or 1; r is 0 or 1; X¹ is CH₂, O or NH;

represents the point of attachment of the linker to the drug; and *represents the point of attachment to the remainder of the linker.

In one embodiment, the linker comprises a segment according tostructural formula (VIIIa), (VIIIb), or (VIIIc):

or a hydrolyzed derivative thereof, wherein: R^(q) is H orO—(CH₂CH₂O)₁₁—CH₃; x is 0 or 1; y is 0 or 1; G³ is —CH₂CH₂CH₂SO₃H or—CH₂CH₂O—(CH₂CH₂O)₁₁—CH₃; R^(w) is —O—CH₂CH₂SO₃H or—NH(CO)—CH₂CH₂O—(CH₂CH₂O)₁₂—CH₃; * represents the point of attachment tothe remainder of the linker; and

represents the point of attachment of the linker to the antibody.

In one embodiment, the linker comprises a polyethylene glycol segmenthaving from 1 to 6 ethylene glycol units.

In one embodiment, m is 2, 3 or 4.

In one embodiment, linker L is selected from IVa or IVb.

In one embodiment, linker L is selected from the group consisting ofIVa.1-IVa.8, IVb-1-IVb.19, IVc.1-IVc.7, Ivd.1-IVd.4, Va.1-Va.12,Vb.1-Vb.10, Vc.1-Vc.11, Vd.1-Vd.6, Ve.1-Ve.2 VIa.1, VIc.1-VIc.2,VId.1-VId.4, VIIa.1-VIIa.4, VIIb.1-VIIb.8, VIIc.1-VIIc.6 in either theclosed or open form.

In one embodiment, the linker L is selected from the group consisting ofIVb.2, IVc.5, IVc.6, IVc.7, IVd.4, Vb.9, VIIa.1, VIIa.3, VIIc.1, VIIc.4,and VIIc.5, wherein the maleimide of each linker has reacted with theantibody Ab, forming a covalent attachment as either a succinimide(closed fora) or succinamide (open form).

In one embodiment, the linker L is selected from the group consisting ofIVb.2, IVc.5, IVc.6, IVd.4, VIIa.1 VIIa.3, VIIc.1, VIIc.4, VIIc.5,wherein the maleimide of each linker has reacted with the antibody Ab,forming a covalent attachment as either a succinimide (closed form) orsuccinamide (open form).

In one embodiment, the linker L is selected from the group consisting ofIVb.2, VIIa.3, IVc.6, and VIIc.1, wherein

is the attachment point to drug D and @ is the attachment point to theLK, wherein when the linker is in the open form as shown below, @ can beeither at the α-position or β-position of the carboxylic acid next toit:

In one embodiment, LK is a linkage formed with an amino group on theanti-hB7H3 antibody Ab.

In one embodiment, LK is an amide or a thiourea.

In one embodiment, LK is a linkage formed with a sulfhydryl group on theanti-hB7-H3 antibody Ab.

In one embodiment, LK is a thioether.

In one embodiment, LK is selected from the group consisting of amide,thiourea and thioether; and in is an integer ranging from 1 to 8.

In one embodiment, 1) is a Bcl-xL inhibitor as described herein; L isselected from the group consisting of linkers IVa.1-IVa.8, IVb.1-IVb.19,IVc.1-IVc.7, IVd.1-IVd.4, Va.1-Va.12, Vb.1-Vb.10, Vc.1-Vc.11, Vd.1-Vd0.6, Ve.1-Ve.2, VIa.1, VIc.1-VIc.2, VId.1-VId.4, VIIa.1-VIIa.4,VIIb.1-VIIb.8, and VIIc.1-VIIc.6, wherein each linker has reacted withthe antibody, Ab, forming a covalent attachment; LK is thioether; and mis an integer ranging from 1 to 8.

In one embodiment, D is the Bcl-xL inhibitor selected from the groupconsisting of the following compounds modified in that the hydrogencorresponding to the 4 position of structural formula (IIa), (IIb),(IIc), or (IId) is not present, forming a monoradical;

-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxyl}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[(2-carboxyethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   1-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]amino}-1,2-dideoxy-D-arabino-hexitol;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(H)-yl]-3-(1-{[3-(2-{[3-hydroxy-2-(hydroxymethyl)propyl]amino}ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylic    acid; and-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3-(2-{[(3S)-3,4-dihydroxybutyl]amino}ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylic    acid;

L is selected from the group consisting of linkers IVb.2, IVc.5, IVc.6,IVc.7, IVd.4, Vb.9, Vc.11, VIIa.1, VIIa.3, VIIc.1, VIIc.4, and VIIc.5 ineither closed or open forms;

LK is thioether; and

m is an integer ranging from 2 to 4.

In one embodiment, the ADC is selected from the group consisting offormulae i-vi:

wherein m is an integer from 1 to 6. In one embodiment, Ab is ananti-hB7-H3 antibody, wherein the anti-hB7-H3 antibody comprises a heavychain CDR3 domain comprising the amino acid sequence set forth in SEQ IDNO: 35, a heavy chain CDR2 domain comprising the amino acid sequence setforth in SEQ ID NO: 34, and a heavy chain CDR1 domain comprising theamino acid sequence set forth in SEQ ID NO: 33, and a light chain CDR3domain comprising the amino acid sequence set forth in SEQ ID NO: 39, alight chain CDR2 domain comprising the amino acid sequence set forth inSEQ ID NO: 38, and a light chain CDR1 domain comprising the amino acidsequence set forth in SEQ ID NO: 37. In one embodiment, the Ab is ananti-hB7-H3 antibody, wherein the anti-hB7H3 antibody comprises a heavychain variable region comprising the amino acid sequence set forth inSEQ ID NO: 147, and a light chain variable region comprising the aminoacid sequence set forth in SEQ ID NO: 144. In one embodiment, Ab is ananti-hB7-H3 antibody, wherein the anti-hB7-H3 antibody comprises a heavychain constant region comprising the amino acid sequence set forth inSEQ ID NO: 160 and/or a light chain constant region comprising the aminoacid sequence set forth in SEQ ID NO: 161. In one embodiment, Ab is ananti-hB7-H3 antibody, wherein the anti-hB7-H3 antibody comprises a heavychain comprising the amino acid sequence set forth in SEQ ID NO: 168,and a light chain comprising the amino acid sequence set forth in SEQ IDNO: 169. In one embodiment, Ab is an anti-hB7-H3 antibody, wherein theanti-hB7-H3 antibody comprises a heavy chain CDR3 domain comprising theamino acid sequence set forth in SEQ ID NO: 12, a heavy chain CDR2domain comprising the amino acid sequence set forth in SEQ ID NO: 140,and a heavy chain CDR1 domain comprising the amino acid sequence setforth in SEQ ID NO: 10; and a light chain CDR3 domain comprising theamino acid sequence set forth in SEQ ID NO: 15, a light chain CDR2domain comprising the amino acid sequence set forth in SEQ ID NO: 7, anda light chain CDR1 domain comprising the amino acid sequence set forthin SEQ ID NO: 136. In one embodiment, the Ab is an anti-hB7-H3 antibody,wherein the anti-hB7H3 antibody comprises a heavy chain variable regioncomprising the amino acid sequence set forth in SEQ ID NO: 139, and alight chain variable region comprising the amino acid sequence set forthin SEQ ID NO: 135. In one embodiment, Ab is an anti-hB7-H3 antibody,wherein the anti-hB7-H3 antibody comprises a heavy chain constant regioncomprising the amino acid sequence set forth in SEQ ID NO: 160 and/or alight chain constant region comprising the amino acid sequence set forthin SEQ ID NO: 161. In one embodiment, Ab is an anti-hB7-H3 antibody,wherein the anti-hB7-H3 antibody comprises a heavy chain comprising theamino acid sequence set forth in SEQ ID NO: 170, and a light chaincomprising the amino acid sequence set forth in SEQ ID NO: 171.

In one embodiment, m is an integer from 2 to 6. In one embodiment, m is2.

In one embodiment, the ADC comprises an anti-hB7-H3 antibody comprisinga heavy chain CDR3 domain comprising the amino acid sequence set forthin SEQ ID NO: 12, a heavy chain CDR2 domain comprising the amino acidsequence set forth in SEQ ID NO: 140, and a heavy chain CDR1 domaincomprising the amino acid sequence set forth in SEQ ID NO: 10; a lightchain CDR3 domain comprising the amino acid sequence set forth in SEQ IDNO: 15, a light chain CDR2 domain comprising the amino acid sequence setforth in SEQ ID NO: 7, and a light chain CDR1 domain comprising theamino acid sequence set forth in SEQ ID NO: 136 or 138.

In one embodiment, the ADC comprises an antibody comprising a heavychain variable region comprising the amino acid sequence set forth inSEQ ID NO: 139, and a light chain variable region comprising the aminoacid sequence set forth in SEQ ID NO: 135.

In one embodiment, the ADC comprises an the antibody comprising a heavychain variable region comprising the amino acid sequence set forth inSEQ ID NO: 139, and a light chain variable region comprising the aminoacid sequence set forth in SEQ ID NO: 137.

In one embodiment, the ADC comprises an antibody comprising a lightchain CDR3 domain comprising the amino acid sequence set forth in SEQ IDNO: 39, a light chain CDR2 domain comprising the amino acid sequence setforth in SEQ ID NO: 38, and a light chain CDR1 domain comprising theamino acid sequence set forth in SEQ ID NO: 37; and a heavy chain CDR3domain comprising the amino acid sequence set forth in SEQ ID NO: 35, aheavy chain CDR2 domain comprising the amino acid sequence set forth inSEQ ID NO: 34, and a heavy chain CDR1 domain comprising the amino acidsequence set forth in SEQ ID NO: 33.

In one embodiment, the ADC comprises an antibody comprising a heavychain variable region comprising the amino acid sequence set forth inSEQ ID NO: 147, and a light chain variable region comprising the aminoacid sequence set forth in SEQ ID NO: 144.

In one embodiment, the ADC is selected from the group consisting ofhuAb3v2.5-CZ, huAb3v2.5-TX, huAb3v2.5-TV, huAb3v2.5-YY, huAb3v2.5-AAA,huAb3v2.5-AAD, huAb3v2.6-CZ, huAb3v2.6-TX, huAb3v2.6-TV, huAb3v2.6-YY,huAb3v2.6-AAD, huAb13v1-CZ, huAb13v1-TX, huAb13v1-TV, huAb13v1-YY,huAb13v1-AAA, huAb13-v1-AAD, wherein CZ, TX, TV, YY, AAA, and AAD aresynthons disclosed in Table B, and wherein the conjugated synthons areeither in open or closed form.

In one aspect, the present invention provides a pharmaceuticalcomposition comprising an effective amount of an ADC described herein,and a pharmaceutically acceptable carrier.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising an ADC mixture comprising a plurality of ADCsdescribed herein, and a pharmaceutically acceptable carrier.

In one embodiment, the ADC mixture has an average drug to antibody ratio(DAR) of 1.5 to 4.

In one embodiment, the ADC mixture comprises ADCs each having a DAR of1.5 to 8.

In one aspect, the present invention provides a method for treatingcancer, comprising administering a therapeutically effective amount ofan ADC described herein to a subject in need thereof.

In one embodiment, the cancer is selected from the group consisting ofsmall cell lung cancer, non small cell lung cancer, breast cancer,ovarian cancer, a glioblastoma, prostate cancer, pancreatic cancer,colon cancer, gastric cancer, melanoma, hepatocellular carcinoma, headand neck cancer, kidney cancer, leukemia, e.g., acute myeloid leukemia(AML), and lymphoma, e.g., non-Hodgkin's lymphoma (NHL).

In one embodiment, the cancer is a squamous cell carcinoma. In oneembodiment, the squamous cell carcinoma is squamous lung cancer orsquamous head and neck cancer.

In one embodiment, the cancer is triple negative breast cancer.

In one embodiment, the cancer is non-small cell lung cancer.

In one embodiment, the cancer is characterized as having an activatingEGFR mutation. In one embodiment, the activating EGFR mutation isselected from the group consisting of an exon 19 deletion mutation, asingle-point substitution mutation L858R in exon 21, a T790M pointmutation, and combinations thereof.

In one aspect, the present invention provides a method for inhibiting ordecreasing solid tumor growth in a subject having a solid tumor, saidmethod comprising administering an effective amount of an ADC describedherein to the subject having the solid tumor, such that the solid tumorgrowth is inhibited or decreased.

In one embodiment, the solid tumor is a non-small cell lung carcinoma.

In one embodiment, the ADC is administered in combination with anadditional agent or an additional therapy.

In one embodiment, the additional agent is selected from the groupconsisting of an anti-PD1 antibody (e.g. pembrolizumab), an anti-PD-L1antibody (e.g., atezolizumab), an anti-CTLA-4 antibody (e.g.ipilimumab), a MEK inhibitor (e.g. trametinib), an ERK inhibitor, a BRAEinhibitor (e.g. dabrafenib), osimertinib, erlotinib, gefitinib,sorafenib, a CDK9 inhibitor (e.g. dinaciclib), a MCL-1 inhibitor,temozolomide, a Bcl-2 inhibitor (e.g. venetoclax), a Bcl-xL inhibitor,ibrutinib, a mTOR inhibitor (e.g. everolimus), a PI3K inhibitor (e.g.buparlisib), duvelisib, idelalisib, an AKT inhibitor, a HER2 inhibitor(e.g. lapatinib), a taxane (e.g. docetaxel, paclitaxel, nab-paclitaxel),an ADC comprising an auristatin, an ADC comprising a PBD (e.g.rovalpituzumab tesirine), an ADC comprising a maytansinoid (e.g. TDM1),a TRAIL agonist, a proteasome inhibitor (e.g. bortezomib), and anicotinamide phosphoribosyltransferase (NAMPT) inhibitor.

In one embodiment, the anti-B7-H3 ADCs of the invention are administeredin combination with venetoclax to a human subject for the treatment ofsmall cell lung cancer (SCLC).

In one embodiment, the additional therapy is radiation.

In one embodiment, the additional agent is a chemotherapeutic agent.

In one aspect, the present invention provides a process for thepreparation of an ADC according to structural formula (I):

(D-L-LK_(m)Ab  (I)

wherein:

D is the Bcl-xL inhibitor drug of formula (IIa), (IIb), (IIc), or (IId)as disclosed herein;

L is the linker as disclosed herein;

Ab is an hB7-H3 antibody, wherein the hB7-H3 antibody comprises theheavy and light chain CDRs of huAb3v2.5, huAb3v2.6, or huAb1.3v1;

LK represents a covalent linkage linking linker L to antibody Ab; and

m is an integer ranging from 1 to 20;

the process comprising:

treating an antibody in an aqueous solution with an effective amount ofa disulfide reducing agent at 30-40° C. for at least 15 minutes, andthen cooling the antibody solution to 20-27° C.;

adding to the reduced antibody solution a solution of water/dimethylsulfoxide comprising a synthon selected from the group of 2.1 to 2.176(Table B);

adjusting the pH of the solution to a pH of 7.5 to 8.5;

allowing the reaction to run for 48 to 80 hours to form the ADC;

wherein the mass is shifted by 18±2 amu for each hydrolysis of asuccinimide to a succinimide as measured by electron spray massspectrometry; and

wherein the ADC is optionally purified by hydrophobic interactionchromatography.

In one embodiment, m is 2.

In another aspect, the present invention provides an ADC prepared by theprocess as described above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical representation of the epitope grouping of murineanti-B7-H3 hybridoma antibodies as determined by pair-wise bindingassays.

FIG. 2 depicts an antibody reduction, modification with a maleimidederivative to give a thiosuccinimide intermediate, and subsequenthydrolysis of thiosuccinimide moiety

FIG. 3 depicts the structure of anantibody-maleimidocaproyl-vc-PABA-MMAE ADC.

FIG. 4 depicts the structure of a PBD dimer (SGD-1882) conjugated to anantibody (Ab) via a maleimidocaproyl-valine-alanine linker (collectivelyreferred to as SGD-1910).

FIG. 5 depicts the MS characterization of light chain and heavy chain ofhuAb13v1 1) prior to conjugation, 2) after conjugation to a maleimidederivative to give a thiosuccinimide intermediate and 3) post pH8-mediated hydrolysis of the thiosuccinimide ring.

DETAILED DESCRIPTION OF THE INVENTION

Various aspects of the invention relate to anti-B7-H3 antibodies andantibody fragments, anti-B7-H3 ADCs, and pharmaceutical compositionsthereof, as well as nucleic acids, recombinant expression vectors andhost cells for making such antibodies and fragments. Methods of usingthe antibodies, fragments, and ADCs described herein to detect humanB7-H3, to inhibit human B7-H3 activity (in vitro or in vivo), and totreat cancers are also encompassed by the invention. In certainembodiments, the invention provides anti-B7-H3 ADCs, including ADCscomprising Bcl-xL inhibitors, synthons useful for synthesizing the ADCs,compositions comprising the ADCs, methods of making the ADCs, andvarious methods of using the ADCs.

As will be appreciated by skilled artisans, the ADCs disclosed hereinare “modular” in nature. Throughout the instant disclosure, variousspecific embodiments of the various “modules” comprising the ADCs, aswell as the synthons useful for synthesizing the ADCs, are described. Asspecific non-limiting examples, specific embodiments of antibodies,linkers, and Bcl-xL inhibitors that may comprise the ADCs and synthonsare described. It is intended that all of the specific embodimentsdescribed may be combined with each other as though each specificcombination were explicitly described individually.

It will also be appreciated by skilled artisans that the various ADCsand/or ADC synthons described herein may be in the form of salts, and incertain embodiments, particularly pharmaceutically acceptable salts. Thecompounds of the present disclosure that possess a sufficiently acidic,a sufficiently basic, or both functional groups, can react with any of anumber of inorganic bases, and inorganic and organic acids, to form asalt. Alternatively, compounds that are inherently charged, such asthose with a quaternary nitrogen, can form a salt with an appropriatecounterion, e.g., a halide such as a bromide, chloride, or fluoride.

Acids commonly employed to form acid addition salts are inorganic acidssuch as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuricacid, phosphoric acid, and the like, and organic acids such asp-toluenesulfonic acid, methanesulfonic acid, oxalic acid,p-bromophenyl-sulfonic acid, carbonic acid, succinic acid, citric acid,etc. Base addition salts include those derived from inorganic bases,such as ammonium and alkali or alkaline earth metal hydroxides,carbonates, bicarbonates, and the like.

In the disclosure below, if both structural diagrams and nomenclatureare included and if the nomenclature conflicts with the structuraldiagram, the structural diagram controls.

An outline of the Detailed Description of the Invention is providedbelow:

I. Definitions II. Anti-B7-H3 Antibodies

II. A. Anti-B7-H3 Chimeric Antibodies

II.B. Humanized Anti-B7-H3 Antibodies

III. Anti-B7-H3 Antibody Drug Conjugates (ADCs)

III.A. Anti-B7-H3/Bcl-xL Inhibitor ADCs

-   -   III.A.1. Bcl-xL Inhibitors    -   III.A.2 Bcl-xL Linkers        -   Cleavable Linkers        -   Non-Cleavable Linkers        -   Groups Used to Attach Linkers to Anti-B7-H3 Antibodies        -   Linker Selection Considerations    -   III.A.3. Bcl-xL ADC Synthons    -   III.A.4 Methods of Synthesis of Bcl-xL ADCs    -   III.A.5. General Methods for Synthesizing Bcl-xL, Inhibitors    -   III.A.6. General Methods for Synthesizing Synthons    -   III.A.7. General Methods for Synthesizing Anti-B7-H3 ADCs

III.B. Anti-B7-H3 ADCs: Other Exemplary Drugs for Conjugation

III.C. Anti-B7-H3 ADCs: Other Exemplary Linkers

IV. Purification of Anti-B7-H3 ADCs V. Uses of Anti-B7-H3 Antibodies andAnti-B7-H3 ADCs VI. Pharmaceutical Compositions I. Definitions

In order that the invention may be more readily understood, certainterms are first defined. In addition, it should be noted that whenever avalue or range of values of a parameter are recited, it is intended thatvalues and ranges intermediate to the recited values are also intendedto be part of this invention.

The term “anti-B7-H3 antibody” refers to an antibody that specificallybinds to B7-H3. An antibody “which binds” an antigen of interest, i.e.,B7-H3, is one capable of binding that antigen with sufficient affinitysuch that the antibody is useful in targeting a cell expressing theantigen. In a preferred embodiment, the antibody specifically binds tohuman B7-H3 (hB7-H3). Examples of anti-B7-H3 antibodies are disclosed inthe examples below. Unless otherwise indicated, the term “anti-B7-H3antibody” is meant to refer to an antibody which binds to wild typeB7-H3 (e.g., a 4IgB7-H3 isoform of B7-H3) or any variant of B7-H3. Theamino acid sequence of wild type human B7-H3 is provided below as SEQ IDNO: 149, where the signal peptide (amino acid residues 1-28) isunderlined.

(SEQ ID NO: 149) MLRRRGSPGMGVHVGAALGALWFCLTGALEVQVPEDPVVALVGTDATLCCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYQGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSILRVVLGANGTYSCLVRNPVLQQDAHSSVTITPQRSPTGAVEVQVPEDPVVALVGTDATLRCSFSPEPGESLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYRGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQPMTFPPEALWVTVGLSVCLIALLVALAFVCWRKIKQSCEEENAGAEDQDGEGEGSKTAL QPLKHSDSKEDDGQEIAThus, in one embodiment of the invention, the antibody or ADC bindshuman B7-H3 as defined in SEQ ID NO: 149. The extracellular domain (ECD)of human B7-H3 is provided in SEQ ID NO: 152 (inclusive of a His tag).As such, in one embodiment of the invention, the antibody of ADC bindsthe ECD of human B7-H3 as described in the ECD of SEQ ID NO: 152.

The terms “specific binding” or “specifically binding”, as used herein,in reference to the interaction of an antibody or an ADC with a secondchemical species, mean that the interaction is dependent upon thepresence of a particular structure (e.g., an antigenic determinant orepitope) on the chemical species; for example, an antibody recognizesand binds to a specific protein structure rather than to proteinsgenerally. If an antibody or ADC is specific for epitope “A”, thepresence of a molecule containing epitope A (or free, unlabeled A), in areaction containing labeled “A” and the antibody, will reduce the amountof labeled A bound to the antibody or ADC. By way of example, anantibody “binds specifically” to a target if the antibody, when labeled,can be competed away from its target by the corresponding non-labeledantibody. In one embodiment, an antibody specifically binds to a target,e.g., B7-H3, if the antibody has a K_(D) for the target of at leastabout 10⁻⁴ M, 10⁻⁵ M, 10⁻⁶ M, 10-7 M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M,10⁻¹² M, or less (less meaning a number that is less than 10⁻¹², e.g.10⁻¹³). In one embodiment, the term “specific binding to B7-H3” or“specifically hinds to B7-H3,” as used herein, refers to an antibody oran ADC that binds to B7-H3 and has a dissociation constant (K_(D)) of1.0×10⁻⁷ M or less, as determined by surface plasmon resonance. It shallbe understood, however, that the antibody or ADC may be capable ofspecifically binding to two or more antigens which are related insequence. For example, in one embodiment, an antibody can specificallybind to both human and a non-human (e.g., mouse or non-human primate)orthologs of B7-H3.

The term “antibody” refers to an immunoglobulin molecule thatspecifically binds to an antigen and comprises a heavy (H) chain(s) anda light (L chain(s). Each heavy chain is comprised of a heavy chainvariable region (abbreviated herein as HCVR or VH) and a heavy chainconstant region. The heavy chain constant region is comprised of threedomains, CH1, CH2 and CH3. Each light chain is comprised of a lightchain variable region (abbreviated herein as LCVR or VL) and a lightchain constant region. The light chain constant region is comprised ofone domain, CL. The VII and VL regions can be further subdivided intoregions of hypervariability, termed complementarity determining regions(CDR), interspersed with regions that are more conserved, termedframework regions (FR). Each VH and VL is composed of three CDRs andfour FRs, arranged from amino-terminus to carboxy-terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. An antibody canbe of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY) and class (e.g.,IgG1, IgG2, IgG 3, IgG4, IgA1 and IgA2) or subclass.

While the term “antibody” is not intended to include antigen bindingportions of an antibody (defined below), it is intended, in certainembodiments, to include a small number of amino acid deletions from thecarboxy end of the heavy chain(s). In one embodiment, an antibodycomprises a heavy chain having 1-5 amino acid deletions the carboxy endof the heavy chain. In one embodiment, an antibody is a monoclonalantibody which is an IgG, having four polypeptide chains, two heavy (H)chains, and two light (L chains) that can bind to hB7-H3. In oneembodiment, an antibody is a monoclonal IgG antibody comprising a lambdaor a kappa light chain.

The term “antigen binding portion” or “antigen binding fragment” of anantibody (or simply “antibody portion” or “antibody fragment”), as usedherein, refers to one or more fragments of an antibody that retain theability to specifically bind to an antigen (e.g., hB7-H3). It has beenshown that the antigen binding function of an antibody can be performedby fragments of a full-length antibody. Such antibody embodiments mayalso be bispecific, dual specific, or multi-specific formats;specifically binding to two or more different antigens. Examples ofbinding fragments encompassed within the term “antigen binding portion”of an antibody include (i) a Fab fragment, a monovalent fragmentconsisting of the VL VH, CL and CHI domains; (ii) a F(ab′)₂ fragment, abivalent fragment comprising two Fab fragments linked by a disulfidebridge at the hinge region; (iii) a Fd fragment consisting of the VH andall domains; (iv) a Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, (v) a dAb fragment (Ward et al., (1989)Nature 341:544-546, Winter et al., PCT publication WO 90/05144 A1 hereinincorporated by reference), which comprises a single variable domain;and (vi) an isolated complementarily determining region (CDR).Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by a synthetic linker that enables them to be made as a singleprotein chain in which the VL and VH regions pair to form monovalentmolecules (known as single chain FVv (scFV), see e.g., Bird et al.(1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad.Sci. USA 85:5879-5883). Such single chain antibodies are also intendedto be encompassed within the term “antigen binding portion” of anantibody. In certain embodiments of the invention, scFv molecules may beincorporated into a fusion protein. Other forms of single chainantibodies, such as diabodies are also encompassed. Diabodies arebivalent, bispecific antibodies in which VH and VL domains are expressedon a single polypeptide chain, but using a linker that is too short toallow for pairing between the two domains on the same chain, therebyforcing the domains to pair with complementary domains of another chainand creating two antigen binding sites (see e.g., Holliger, P., et al.(1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al.(1994) Structure 2:1121-1123). Such antibody binding portions are knownin the art (Kontermann and Dubel eds., Antibody Engineering (2001)Springer-Verlag, New York. 790 pp. (ISBN 3-540-41354-5).

An IgG is a class of antibody comprising two heavy chains and two lightchains arranged in a Y-shape. An IgG constant domain refers to a heavyor light chain constant domain. Exemplary, human IgG heavy chain andlight chain constant domain amino acid sequences are known in the artand represented below in Table A.

TABLE A Sequences of human IgG heavy chain constantdomains and light chain constant domains Sequence Protein IdentifierSequence Ig gamma-1 SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY constantNO: 159 FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS regionLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Ig gamma-1 SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY constantNO: 160 FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS regionLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK mutant KVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVICVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Ig Kappa SEQ ID RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF constantNO: 161 YPREAKVQWKVDNALQSGNSQESVTEQDSKDS regionTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC Ig Lambda SEQ IDQPKAAPSVTLFPPSSEELQANKATLVCLISDF constant NO: 162YPGAVTVAWKADSSPVKAGVETTTPSKQSNNK region YAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds B7-H3 is substantially free of antibodies that specifically bindantigens other than B7-H3). An isolated antibody that specifically bindsB7-H3 may, however, have cross-reactivity to other antigens, such asB7-H3 molecules from other species. Moreover, an isolated antibody maybe substantially free of other cellular material and/or chemicals.

The term “humanized antibody” refers to antibodies which comprise heavyand light chain variable region sequences from a nonhuman species (e.g.,a mouse) but in which at least a portion of the VH and/or VL sequencehas been altered to be more “human-like”, i.e., more similar to humangermline variable sequences. In particular, the term “humanizedantibody” is an antibody or a variant, derivative, analog or fragmentthereof which immunospecifically binds to an antigen of interest andwhich comprises a framework (FR) region having substantially the aminoacid sequence of a human antibody and a complementary determining region(CDR) having substantially the amino acid sequence of a non-humanantibody. As used herein, the term “substantially” in the context of aCDR refers to a CDR having an amino acid sequence at least 80%,preferably at least 85%, at least 90%, at least 95%, at least 98% or atleast 99% identical to the amino acid sequence of a non-human antibodyCDR. A humanized antibody comprises substantially all of at least one,and typically two, variable domains (Fab, Fab′, F(ab′)₂, FabC, Fv) inwhich all or substantially all of the CDR regions correspond to those ofa non-human immunoglobulin (i.e., donor antibody) and all orsubstantially all of the framework regions are those of a humanimmunoglobulin consensus sequence. Preferably, a humanized antibody alsocomprises at least a portion of an immunoglobulin constant region (Fe),typically that of a human immunoglobulin. In some embodiments, ahumanized antibody contains both the light chain as well as at least thevariable domain of a heavy chain. The antibody also may include the CH1,hinge, CH2, CH3, and CH4 regions of the heavy chain. In someembodiments, a humanized antibody only contains a humanized light chain.In other embodiments, a humanized antibody only contains a humanizedheavy chain. In specific embodiments, a humanized antibody only containsa humanized variable domain of a light chain and/or humanized heavychain.

The humanized antibody can be selected from any class ofimmunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype,including without limitation IgG1, IgG2, IgG3 and IgG4. The humanizedantibody may comprise sequences from more than one class or isotype, andparticular constant domains may be selected to optimize desired effectorfunctions using techniques well-known in the art.

The terms “Kabat numbering,” “Kabat definitions,” and “Kabat labeling”are used interchangeably herein. These terms, which are recognized inthe art, refer to a system of numbering amino acid residues which aremore variable (i.e., hypervariable) than other amino acid residues inthe heavy and light chain variable regions of an antibody, or an antigenbinding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci.190:382-391 and, Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242). For the heavy chainvariable region, the hypervariable region ranges from amino acidpositions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, andamino acid positions 95 to 102 for CDR3. For the light chain variableregion, the hypervariable region ranges from amino acid positions 24 to34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acidpositions 89 to 97 for CDR3.

As used herein, the term “CDR” refers to the complementarity determiningregion within antibody variable sequences. There are three CDRs in eachof the variable regions of the heavy chain (HC) and the light chain(LC), which are designated CDR1, CDR2 and CDR3 (or specifically HC CDR1,HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3), for each of thevariable regions. The term “CDR set” as used herein refers to a group ofthree CDRs that occur in a single variable region capable of binding theantigen. The exact boundaries of these CDRs have been defineddifferently according to different systems. The system described byKabat (Kabat et al., Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md. (1987) and (1991)) notonly provides an unambiguous residue numbering system applicable to anyvariable region of an antibody, but also provides precise residueboundaries defining the three CDRs. These CDRs may be referred to asKabat CDRs. Chothia and coworkers (Chothia & Lesk, J. Mol. Biol.196:901-917 (1987) and Chothia et al., Nature 342:877-883 (1989)) foundthat certain sub-portions within Kabat CDRs adopt nearly identicalpeptide backbone conformations, despite having great diversity at thelevel of amino acid sequence. These sub-portions were designated as L1,L2 and L3 or H1, H2 and H3 where the “L” and the “H” designates thelight chain and the heavy chains regions, respectively. These regionsmay be referred to as Chothia CDRs, which have boundaries that overlapwith Kabat CDRs. Other boundaries defining CDRs overlapping with theKabat CDRs have been described by Padlan (FASEB J. 9:133-139 (1995)) andMacCallum (J. Mol. Biol. 262(5):732-45 (1996)). Still other CDR boundarydefinitions may not strictly follow one of the above systems, but willnonetheless overlap with the Kabat CDRs, although they may be shortenedor lengthened in light of prediction or experimental findings thatparticular residues or groups of residues or even entire CDRs do notsignificantly impact antigen binding. The methods used herein mayutilize CDRs defined according to any of these systems, althoughpreferred embodiments use Kabat or Chothia defined CDRs.

As used herein, the term “framework” or “framework sequence” refers tothe remaining sequences of a variable region minus the CDRs. Because theexact definition of a CDR sequence can be determined by differentsystems, the meaning of a framework sequence is subject tocorrespondingly different interpretations. The six CDRs (CDR-L1, CDR-L2,and CDR-L3 of light chain and CDR-H1, CDR-H2, and CDR-H3 of heavy chain)also divide the framework regions on the light chain and the heavy chaininto four sub-regions (FR1, FR2, FR3 and FR4) on each chain, in whichCDR1 is positioned between FR1, and FR2, CDR2 between FR2 and FR3, andCDR3 between FR3 and FR4. Without specifying the particular sub-regionsas FR1, FR2, FR3 or FR4, a framework region, as referred by others,represents the combined FR's within the variable region of a single,naturally occurring immunoglobulin chain. As used herein, a FRrepresents one of the four sub-regions, and FRs represents two or moreof the four sub-regions constituting a framework region.

The framework and CDR regions of a humanized antibody need notcorrespond precisely to the parental sequences, e.g., the donor antibodyCDR or the consensus framework may be mutagenized by substitution,insertion and/or deletion of at least one amino acid residue so that theCDR or framework residue at that site does not correspond to either thedonor antibody or the consensus framework. In a preferred embodiment,such mutations, however, will not be extensive. Usually, at least 80%,preferably at least 85%, more preferably at least 90%, and mostpreferably at least 95% of the humanized antibody residues willcorrespond to those of the parental FR and CDR sequences. As usedherein, the term “consensus framework” refers to the framework region inthe consensus immunoglobulin sequence. As used herein, the term“consensus immunoglobulin sequence” refers to the sequence formed fromthe most frequently occurring amino acids (or nucleotides) in a familyof related immunoglobulin sequences (See e.g., Winnaker, From Genes toClones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family ofimmunoglobulins, each position in the consensus sequence is occupied bythe amino acid occurring most frequently at that position in the family.If two amino acids occur equally frequently, either can be included inthe consensus sequence.

The term “human acceptor framework”, as used herein, is meant to referto a framework of an antibody or antibody fragment thereof comprisingthe amino acid sequence of a VH or VL framework derived from a humanantibody or antibody fragment thereof or a human consensus sequenceframework into which CDR's from a non-human species may be incorporated.

“Percent (%) amino acid sequence identity” with respect to a peptide orpolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the specific peptide or polypeptide sequence, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN orMegalign (DNASTAR) software. Those skilled in the art can determineappropriate parameters for measuring alignment, including any algorithmsneeded to achieve maximal alignment over the full length of thesequences being compared. In one embodiment, the invention includes anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to an amino acid sequence set forth in any one of SEQ ID NOs: 1to 148.

The term “multivalent antibody” is used herein to denote an antibodycomprising two or more antigen binding sites. In certain embodiments,the multivalent antibody may be engineered to have the three or moreantigen binding sites, and is generally not a naturally occurringantibody.

The term “multispecific antibody” refers to an antibody capable ofbinding two or more unrelated antigens. In one embodiment, themultispecific antibody is a bispecific antibody that is capable ofbinding to two unrelated antigens, e.g., a bispecific antibody, orantigen-binding portion thereof, that binds B7-H3 and CD3.

The term “dual variable domain” or “DVD,” as used interchangeablyherein, are antigen binding proteins that comprise two or more antigenbinding sites and are tetravalent or multivalent binding proteins. SuchDVDs may be monospecific, i.e., capable of binding one antigen ormultispecific, i.e. capable of binding two or more antigens. DVD bindingproteins comprising two heavy chain DVD polypeptides and two light chainDVD polypeptides are referred to a DVD Ig. Each half of a DVD Igcomprises a heavy chain DVD polypeptide, and a light chain DVDpolypeptide, and two antigen binding sites. Each binding site comprisesa heavy chain variable domain and a light chain variable domain with atotal of 6 CDRs involved in antigen binding per antigen binding site. Inone embodiment, the CDRs described herein are used in an anti-B7-H3 DVD.

The term “chimeric antigen receptor” or “CAR” refers to a recombinantprotein comprising at least (1) an antigen-binding region, e.g., avariable heavy or light chain of an antibody, (2) a transmembrane domainto anchor the CAR into a T cell, and (3) one or more intracellularsignaling domains.

The term “activity” includes activities such as the bindingspecificity/affinity of an antibody or ADC for an antigen, for example,an anti-hB7-H3 antibody that binds to an hB7-H3 antigen and/or theneutralizing potency of an antibody, for example, an anti-hB7-H3antibody whose binding to hB7-H3 inhibits the biological activity ofhB7-H3, e.g., inhibition of proliferation of B7-H3 expressing celllines, e.g., human H1146 lung carcinoma cells, human H1650 lungcarcinoma cells, or human EBC1 lung carcinoma cells.

The term “non small-cell lung carcinoma (NSCLC) xenograft assay,” asused herein, refers to an in vivo assay used to determine whether ananti-B7-H3 antibody or ADC, can inhibit tumor growth (e.g., furthergrowth) and/or decrease tumor growth resulting from the transplantationof NSCLC cells into an immunodeficient mouse. An NSCLC xenograft assayincludes transplantation of NSCLC cells into an immunodeficient mousesuch that a tumor grows to a desired size, e.g., 200-250 mm³, whereuponthe antibody or ADC is administered to the mouse to determine whetherthe antibody or ADC can inhibit and/or decrease tumor growth. In certainembodiments, the activity of the antibody or ADC is determined accordingto the percent tumor growth inhibition (% TGI) relative to a controlantibody, e.g., a human IgG antibody (or collection thereof) which doesnot specifically bind tumor cells, e.g., is directed to an antigen notassociated with cancer or is obtained from a source which isnoncancerous (e.g., normal human serum). In such embodiments, theantibody (or ADC) and the control antibody are administered to the mouseat the same dose, with the same frequency, and via the same route. Inone embodiment, the mouse used in the NSCLC xenograft assay is a severecombined immunodeficiency (SCID) mouse and/or an athymic CD-1 nudemouse. Examples of NSCLC cells that may be used in the NSCLC xenograftassay include, but are not limited to, H1299 cells (NCI-H1299 [H-1299](ATCC® CRL-5803)), H1975 cells (NCI-H1975 cells [H1975] (ATCC®CRL-5908™)), and EBC-1 cells.

The term “small-cell lung carcinoma (SCLC) xenograft assay,” as usedherein, refers to an in vivo assay used to determine whether ananti-B7-H3 antibody or ADC, can inhibit tumor growth (e.g., furthergrowth) and/or decrease tumor growth resulting from the transplantationof SCLC cells into an immunodeficient mouse. An SCLC xenograft assayincludes transplantation of SCLC cells into an immunodeficient mousesuch that a tumor grows to a desired size, e.g., 200-250 mm³, whereuponthe antibody or ADC is administered to the mouse to determine whetherthe antibody or ADC can inhibit and/or decrease tumor growth. In certainembodiments, the activity of the antibody or ADC is determined accordingto the percent tumor growth inhibition (% TGI) relative to a controlantibody, e.g., a human IgG antibody (or collection thereof) which doesnot specifically bind tumor cells, e.g., is directed to an antigen notassociated with cancer or is obtained from a source which isnoncancerous (e.g., normal human serum). In such embodiments, theantibody (or ADC) and the control antibody are administered to the mouseat the same dose, with the same frequency, and via the same route. Inone embodiment, the mouse used in the NSCLC xenograft assay is a severecombined immunodeficiency (SCID) mouse and/or an athymic CD-1 nudemouse. Examples of SCLC cells that may be used in the SCLC xenograftassay include, but are not limited to, H146 cells (NCI-H146 cells [H146](ATCC® HTB-173™)), and H847 cells (NCI-H-847 [H847] (ATCC® CRL-5846™)).The term “epitope” refers to a region of an antigen that is bound by anantibody or ADC. In certain embodiments, epitope determinants includechemically active surface groupings of molecules such as amino acids,sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments,may have specific three dimensional structural characteristics, and/orspecific charge characteristics. In certain embodiments, an antibody issaid to specifically bind an antigen when it preferentially recognizesits target antigen in a complex mixture of proteins and/ormacromolecules.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIAcore system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). Forfurther descriptions, see Jönsson, U., et al. (1993) Ann. Biol. Clin.51:19-26; Jönsson, U., et al. (1991) BioTechniques 11:620-627; Johnsson,B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson. B., et al.(1991) Anal. Biochem. 198:268-277. In one embodiment, surface plasmonresonance is determined according to the methods described in Example 2.

The term “k_(on),” or “k_(a)”, as used herein, is intended to refer tothe on rate constant for association of an antibody to the antigen toform the antibody/antigen complex.

The term “k_(off)” or “k_(d)”, as used herein, is intended to refer tothe off rate constant for dissociation of an antibody from theantibody/antigen complex.

The term “K_(D)”, as used herein, is intended to refer to theequilibrium dissociation constant of a particular antibody-antigeninteraction e.g., huAb13 antibody and B7-H3). K_(D) is calculated byk_(a)/k_(d).

The term “competitive binding”, as used herein, refers to a situation inwhich a first antibody competes with a second antibody, for a bindingsite on a third molecule, e.g., an antigen. In one embodiment,competitive binding between two antibodies is determined using FACSanalysis.

The term “competitive binding assay” is an assay used to determinewhether two or more antibodies bind to the same epitope. In oneembodiment, a competitive binding assay is a competition fluorescentactivated cell sorting (FACS) assay which is used to deter nine whethertwo or more antibodies bind to the same epitope by determining whetherthe fluorescent signal of a labeled antibody is reduced due to theintroduction of a non-labeled antibody, where competition for the sameepitope will lower the level of fluorescence.

The term “labeled antibody” as used herein, refers to an antibody, or anantigen binding portion thereof with a label incorporated that providesfor the identification of the binding protein, e.g., an antibody.Preferably, the label is a detectable marker, e.g., incorporation of aradiolabeled amino acid or attachment to a polypeptide of biotinylmoieties that can be detected by marked avidin (e.g., streptavidincontaining a fluorescent marker or enzymatic activity that can bedetected by optical or colorimetric methods). Examples of labels forpolypeptides include, but are not limited to, the following:radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In,¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, or ¹⁵³Sm); fluorescent labels (e.g., FITC,rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradishperoxidase, luciferase, alkaline phosphatase); chemiluminescent markers;biotinyl groups; predetermined polypeptide epitopes recognized by asecondary reporter (e.g., leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags); andmagnetic agents, such as gadolinium chelates.

The term “antibody-drug-conjugate” or “ADC” refers to a binding protein,such as an antibody or antigen binding fragment thereof, chemicallylinked to one or more chemical drug(s) (also referred to herein asagent(s), warhead(s), and payload(s)) that may optionally be therapeuticor cytotoxic agents. In a preferred embodiment, an ADC includes anantibody, a drug, (e.g a cytotoxic drug), and a linker that enablesattachment or conjugation of the drug to the antibody. An ADC typicallyhas anywhere from 1 to 8 drugs conjugated to the antibody, includingdrug loaded species of 2, 4, 6, or 8. Non-limiting examples of drugsthat may be included in the ADCs are mitotic inhibitors, antitumorantibiotics, immunomodulating agents, vectors for gene therapy,alkylating agents, antiangiogenic agents, antimetabolites,boron-containing agents, chemoprotective agents, hormones, antihormoneagents, corticosteroids, photoactive therapeutic agents,oligonucleotides, radionuclide agents, topoisomerase inhibitors, kinaseinhibitors (e.g., TEC-family kinase inhibitors and serine/threoninekinase inhibitors), and radiosensitizers. In one embodiment, the drug isa Bcl-xL inhibitor.

The terms “anti-B7-H3 antibody drug conjugate” or “anti-B7-H3 ADC”, usedinterchangeably herein, refer to an ADC comprising an antibody thatspecifically binds to B7-H3, whereby the antibody is conjugated to oneor more chemical agent(s). In one embodiment, the anti-B7-H3 ADCcomprises antibody huAb13v1, huAb3v2.5, or huAb3v2.6 conjugated to anauristatin, e.g., MMAE or MMAF. In one embodiment, the anti-B7-H3 ADCcomprises antibody huAb1.3v1, huAb3v2.5, or huAb3v2.6 conjugated to aBcl-xL inhibitor. In a preferred embodiment, the anti-B7-H3 ADC binds tohuman B7-H3 (hB7-H3).

The term “Bcl-xL inhibitor”, as used herein, refers to a compound whichantagonizes Bcl-xL activity in a cell. In one embodiment, a Bcl-xLinhibitor promotes apoptosis of a cell by inhibiting Bcl-xL activity.

The term “auristatin”, as used herein, refers to a family of antimitoticagents. Auristatin derivatives are also included within the definitionof the term “auristatin”. Examples of auristatins include, but are notlimited to, auristatin E (AE), monomethylauristatin E (MMAE),monomethylauristatin F (MMAF), and synthetic analogs of dolastatin. Inone embodiment, an anti-B7-H3 antibody described herein is conjugated toan auristatin to form an anti-B7-H3 ADC.

As used herein, the term “Ab-vcMMAE” is used to refer to an ADCcomprising an antibody conjugated to monomethylauristatin E (MMAE) via amaleimidocaproyl valine citrulline p-aminobenzyloxycarbamyl (PABA)linker.

As used herein, the term “mcMMAF” is used to refer to a linker/drugcombination of maleimidocaproyl-monomethylauristatin F (MMAF).

The term “drug-to-antibody ratio” or “DAR” refers to the number ofdrugs, e.g., Bcl-xL inhibitor, attached to the antibody of the ADC. TheDAR of an ADC can range from 1 to 8, although higher loads, e.g., 20,are also possible depending on the number of linkage site on anantibody. The term DAR may be used in reference to the number of drugsloaded onto an individual antibody, or, alternatively, may be used inreference to the average or mean DAR of a group of ADCs.

The term “undesired ADC species”, as used herein, refers to any drugloaded species which is to be separated from an ADC species having adifferent drug load. In one embodiment, the term undesired ADC speciesmay refer to drug loaded species of 6 or more, i.e., ADCs with a DAR of6 or more, including DAR6, DAR7, DAR8, and DAR greater than 8 (i.e.,drug loaded species of 6, 7, 8, or greater than 8). In a separateembodiment, the term undesired ADC species may refer to drug loadedspecies of 8 or more, i.e., ADCs with a DAR of 8 or more, includingDAR8, and DAR greater than 8 (i.e., drug loaded species of 8, or greaterthan 8).

The term “ADC mixture”, as used herein, refers to a compositioncontaining a heterogeneous DAR distribution of ADCs. In one embodiment,an ADC mixture contains ADCs having a distribution of DARs of 1 to 8,e.g., 1.5, 2, 4, 6, and 8 (i.e., drug loaded species of 2, 4, 6, and 8).Notably, degradation products may result such that DARs of 1, 3, 5, and7 may also be included in the mixture. Further, ADCs within the mixturemay also have DARs greater than 8. The ADC mixture results frominterchain disulfide reduction followed by conjugation. In oneembodiment, the ADC mixture comprises both ADCs with a DAR of 4 or less(i.e., a drug loaded species of 4 or less) and ADCs with a DAR of 6 ormore (i.e., a drug loaded species of 6 or more).

The term a “xenograft assay”, as used herein, refers to a human tumorxenograft assay, wherein human tumor cells are transplanted, eitherunder the skin or into the organ type in which the tumor originated,into immunocompromised mice that do not reject human cells.

The term “cancer” is meant to refer to or describe the physiologicalcondition in mammals that is typically characterized by unregulated cellgrowth. Examples of cancer include, but are not limited to, carcinoma,lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. Moreparticular examples of such cancers include glioblastoma, acute myeloidleukemia (AML), non-Hodgkin's lymphoma (NHL), non-small cell lungcancer, lung cancer, colon cancer, colorectal cancer, head and neckcancer, breast cancer (e.g., triple negative breast cancer), pancreaticcancer, squamous cell tumors, squamous cell carcinoma (e.g., squamouscell lung cancer or squamous cell head and neck cancer), anal cancer,skin cancer, and vulvar cancer. In one embodiment, the antibodies orADCs of the invention are administered to a patient having a tumor(s)that overexpresses B7-H3. In one embodiment, the antibodies or ADCs ofthe invention are administered to a patient having a solid tumor whichis likely to over-express B7-H3. In one embodiment, the antibodies orADCs of the invention are administered to a patient having squamous cellnon-small cell lung cancer (NSCLC). In one embodiment, the antibodies orADCs of the invention are administered to a patient having solid tumors,including advanced solid tumors. In one embodiment, the antibodies orADCs of the invention are administered to a patient having prostatecancer. In one embodiment, the antibodies or ADCs of the invention areadministered to a patient having non-small cell lung cancer. In oneembodiment, the antibodies or ADCs of the invention are administered toa patient having a glioblastoma. In one embodiment, the antibodies orADCs of the invention are administered to a patient having colon cancer.In one embodiment, the antibodies or ADCs of the invention areadministered to a patient having head and neck cancer. In oneembodiment, the antibodies or ADCs of the invention are administered toa patient having kidney cancer. In one embodiment, the antibodies orADCs of the invention are administered to a patient having clear cellrenal cell carcinoma. In one embodiment, the antibodies or ADCs of theInvention are administered to a patient having glioma. In oneembodiment, the antibodies or ADCs of the invention are administered toa patient having melanoma. In one embodiment, the antibodies or ADCs ofthe invention are administered to a patient having pancreatic cancer. Inone embodiment, the antibodies or ADCs of the invention are administeredto a patient having gastric cancer. In one embodiment, the antibodies orADCs of the invention are administered to a patient having ovariancancer. In one embodiment, the antibodies or ADCs of the invention areadministered to a patient having colorectal cancer. In one embodiment,the antibodies or ADCs of the invention are administered to a patienthaving renal cancer. In one embodiment, the antibodies or ADCs of theinvention are administered to a patient having small cell lung cancer.In one embodiment, the antibodies or ADCs of the invention areadministered to a patient having hepatocellular carcinoma. In oneembodiment, the antibodies or ADCs of the invention are administered toa patient having hypopharyngeal squamous cell carcinoma. In oneembodiment, the antibodies or ADCs of the invention are administered toa patient having neuroblastoma. In one embodiment, the antibodies orADCs of the invention are administered to a patient having breastcancer. In one embodiment, the antibodies or ADCs of the invention areadministered to a patient having endometrial cancer. In one embodiment,the antibodies or ADCs of the invention are administered to a patienthaving urothelial cell carcinoma. In one embodiment, the antibodies orADCs of the invention are administered to a patient having acute myeloidleukemia (AML). In one embodiment, the antibodies or ADCs of theinvention are administered to a patient having non-Hodgkin's lymphoma(NHL).

The term “B7-H3 expressing tumor,” as used herein, refers to a tumorwhich expresses B7-H3 protein. In one embodiment, B7-H3 expression in atumor is determined using immunohistochemical staining of tumor cellmembranes, where any immunohistochemical staining above background levelin a tumor sample indicates that the tumor is a B7-H3 expressing tumor.Methods for detecting expression of B7-H3 in a tumor are known in theart, and include immunohistochemical assays. In contrast, a “B7-H3negative tumor” is defined as a tumor having an absence of B7-H3membrane staining above background in a tumor sample as determined byimmunohistochemical techniques.

The terms “overexpress.” “overexpression,” or “overexpressed”interchangeably refer to a gene that is transcribed or translated at adetectably greater level, usually in a cancer cell, in comparison to anormal cell. Overexpression therefore refers to both overexpression ofprotein and RNA (due to increased transcription, post transcriptionalprocessing, translation, post translational processing, alteredstability, and altered protein degradation), as well as localoverexpression due to altered protein traffic patterns (increasednuclear localization), and augmented functional activity, e.g., as in anincreased enzyme hydrolysis of substrate. Thus, overexpression refers toeither protein or RNA levels. Overexpression can also be by 50%, 60%,70%, 80%, 90% or more in comparison to a normal cell or comparison cell.In certain embodiments, the anti-B7-H3 antibodies or ADCs of theinvention are used to treat solid tumors likely to overexpress B7-H3.

The term “gene amplification”, as used herein, refers to a cellularprocess characterized by the production of multiple copies of anyparticular piece of DNA. For example, a tumor cell may amplify, or copy,chromosomal segments as a result of cell signals and sometimesenvironmental events. The process of gene amplification leads to theproduction of additional copies of the gene. In one embodiment, the geneis B7-H3, i.e., “B7-H3 amplification.” in one embodiment, thecompositions and methods disclosed herein are used to treat a subjecthaving B7-H3 amplified cancer.

The term “administering” as used herein is meant to refer to thedelivery of a substance (e.g., an anti-B7-H3 antibody or ADC) to achievea therapeutic objective (e.g., the treatment of a B7-H3-associateddisorder). Modes of administration may be parenteral, enteral andtopical. Parenteral administration is usually by injection, andincludes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal and intrasternalinjection and infusion.

The term “combination therapy” or “combination” in the context of atherapeutic method (e.g., a treatment method), as used herein, refers tothe administration of two or more therapeutic substances, e.g., ananti-B7-H3 antibody or ADC and an additional therapeutic agent. Theadditional therapeutic agent may be administered concomitant with, priorto, or following the administration of the anti-B7-H3 antibody or ADC.

As used herein, the term “effective amount” or “therapeuticallyeffective amount” refers to the amount of a drug, e.g., an antibody orADC, which is sufficient to reduce or ameliorate the severity and/orduration of a disorder, e.g., cancer, or one or more symptoms thereof,prevent the advancement of a disorder, cause regression of a disorder,prevent the recurrence, development, onset or progression of one or moresymptoms associated with a disorder, detect a disorder, or enhance orimprove the prophylactic or therapeutic effect(s) of another therapy(e.g., prophylactic or therapeutic agent). The effective amount of anantibody or ADC may, for example, inhibit tumor growth (e.g., inhibit anincrease in tumor volume), decrease tumor growth (e.g., decrease tumorvolume), reduce the number of cancer cells, and/or relieve to someextent one or more of the symptoms associated with the cancer. Theeffective amount may, for example, improve disease free survival (DFS),improve overall survival (OS), or decrease likelihood of recurrence.

Various chemical substituents are defined below. In some instances, thenumber of carbon atoms in a substituent (e.g., alkyl, alkanyl, alkenyl,alkynyl, cycloalkyl, heterocyclyl, heteroaryl, and aryl) is indicated bythe prefix “C_(x)-C_(y)” or “C_(x-y)” wherein x is the minimum and y isthe maximum number of carbon atoms. Thus, for example, “C₁-C₆ alkyl”refers to an alkyl containing from 1 to 6 carbon atoms. Illustratingfurther, “C₃-C₈ cycloalkyl” means a saturated hydrocarbyl ringcontaining from 3 to 8 carbon ring atoms. If a substituent is describedas being “substituted,” a hydrogen atom on a carbon or nitrogen isreplaced with a non-hydrogen group. For example, a substituted alkylsubstituent is an alkyl substituent in which at least one hydrogen atomon the alkyl is replaced with a non-hydrogen group. To illustrate,monofluoroalkyl is alkyl substituted with a fluoro radical, anddifluoroalkyl is alkyl substituted with two fluoro radicals. It shouldbe recognized that if there is more than one substitution on asubstituent, each substitution may be identical or different (unlessotherwise stated), If a substituent is described as being “optionallysubstituted”, the substituent may be either (1) not substituted or (2)substituted. Possible substituents include, but are not limited to,C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, aryl, cycloalkyl,heterocyclyl, heteroaryl, halogen, C₁-C₆ haloalkyl, oxo, —CN, NO₂,—OR^(xa), —OC(O)R^(xz), —OC(O)N(R^(xa))₂, —SR^(xa), —S(O)₂R^(xa), —S()₂N(R^(xa))₂, —C(O)R^(xa)—, —C(O)OR^(xa), —C(O)N(R^(xa))₂,—C(O)N(R^(xa))S(O)₂R^(xa), —N(R^(xa))₂, —N(R^(xa))C(O)R^(xz),—N(R^(xa))S(O)₂R^(xz), —N(R^(xa))C(O)O(R^(xz)),—N(R^(xa))C(O)N(R^(xa))₂, —N(R^(xa))S(O)₂N(R^(xa))₂, —(C₁-C₆alkylenyl)-CN, —(C₁-C₆ alkylenyl)-OR^(xa), —(C₁-C₆alkylenyl)-OC(O)R^(xz), —(C₁-C₆ alkylenyl)-OC(O)N(R^(xa))₂, —(C₁-C₆alkylenyl)-SR^(xa), —(C₁-C₆ alkylenyl)-S(O)₂R^(xa), —(C₁-C₆alkylenyl)-S(O)₂N(R^(xa))₂, —(C₁-C₆ alkylenyl)-C(O)R^(xa), —(C₁-C₆alkylenyl)-C(O)OR^(xa), —(C₁-C₆ alkylenyl)-C(O)N(R^(xa))₂, —(C₁-C₆alkylenyl)-C(O)N(R^(xa))S(O)₂R^(xz), —(C₁-C₆ alkylenyl)-N(R^(xa))₂,—(C₁-C₆ alkylenyl)-N(R^(xa))C(O)R^(xz), —(C₁-C₆alkylenyl)-N(R^(xa))S(O)₂R^(xz), —(C₁-C₆alkylenyl)-N(R^(xa))C(O)O(R^(xa)), —(C₁-C₆alkylenyl)-N(R^(xa))C(O)N(R^(xa))₂, or —(C₁-C₆alkylenyl)-N(R^(xa))S(O)₂N(R^(xa))₂; wherein R^(xa), at each occurrence,is independently hydrogen, aryl, cycloalkyl, heterocyclyl, heteroaryl,C₁-C₆ alkyl, or C₁-C₆ haloalkyl; and R^(xz), at each occurrence, isindependently aryl, cycloalkyl, heterocyclyl, heteroaryl, C₁-C₆ alkyl orC₁-C₆ haloalkyl.

Various ADCs, synthons and Bcl-xL, inhibitors comprising the ADCs and/orsynthons are described in some embodiments herein by reference tostructural formulae including substituents. It is to be understood thatthe various groups comprising substituents may be combined as valenceand stability permit. Combinations of substituents and variablesenvisioned by this disclosure are only those that result in theformation of stable compounds. As used herein, the term “stable” refersto compounds that possess stability sufficient to allow manufacture andthat maintain the integrity of the compound for a sufficient period oftime to be useful for the purpose detailed herein.

As used herein, the following terms are intended to have the followingmeanings:

The term “alkoxy” refers to a group of the formula —OR^(xa), whereR^(xa) is an alkyl group. Representative alkoxy groups include methoxy,ethoxy, propoxy, tert-butoxy and the like.

The term “alkoxyalkyl” refers to an alkyl group substituted with analkoxy group and may be represented by the general formula —R^(b)OR^(xa)where R^(b) is an alkylene group and R^(xa) is an alkyl group.

The term “alkyl” by itself or as part of another substituent refers to asaturated or unsaturated branched, straight-chain or cyclic monovalenthydrocarbon radical that is derived by the removal of one hydrogen atomfrom a single carbon atom of a parent alkane, alkene or alkyne. Typicalalkyl groups include, but are not limited to, methyl; ethyls such asethanyl, ethenyl, ethynyl; propyls such as propan-1-yl; propan-2-yl,cyclopropan-1-yl; prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl,cycloprop-1-en-1-yl; cycloprop-2-en-1-yl, prop-1-yn-1-yl prop-2-yn-1-yl,etc.; butyls such as butan-2-yl, 2-methyl-propan-1-yl,2-methyl-propan-2-yl, cyclobutan-1-yl, but-1-en-1-yl, but-1-en-2-yl,2-methyl-prop-1-en-1-yl, but-2-en-1-yl but-2-en-2-yl, beta-1,3-dien-1buta-1,3-dien-2-yl, cyclobut-1-en-1-yl, but-1-yn-1-yl, but-1-yn-3-yl,but-3-yn-1-yl, etc.; and the like. Where specific levels of saturationare intended, the nomenclature “alkanyl,” “alkenyl” and/or “alkynyl” areused, as defined below. The term “lower alkyl” refers to alkyl groupswith 1 to 6 carbons.

The term “alkanyl” by itself or as part of another substituent refers toa saturated branched, straight-chain or cyclic alkyl derived by theremoval of one hydrogen atom from a single carbon atom of a parentalkane. Typical alkanyl groups include, but are not limited to, methyl;ethynyl; propenyls such as propan-1-yl, propan-2-yl (isopropyl),cyclopropan-1-yl, etc.; butanyls such as butan-1-yl, butan-2-yl(sec-butyl), 2-methyl-propan-1-yl(isobutyl), 2-methyl-propan-2-yl(t-butyl), cyclobutan-1-yl, etc.; and the like.

The term “alkenyl” by itself or as part of another substituent refers toan unsaturated branched, straight-chain or cyclic alkyl having at leastone carbon-carbon double bond derived by the removal of one hydrogenatom from a single carbon atom of a parent alkene. Typical alkenylgroups include, but are not limited to, ethenyl propenyls such asprop-1-en-1-yl, prop-1-en-2-yl, prop 2 en 1 yl, prop-2-en-2-yl,cycloprop-1-en-1-0; cycloprop-2-en-1-yl; butenyls such as but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,buta-1,3-dien-1-yl, buta-1,3-dien-2-yl; cyclobut-1-en-1-yl,cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, etc.; and the like.

The term “alkynyl” by itself or as part of another substituent refers toan unsaturated branched, straight-chain or cyclic alkyl having at leastone carbon-carbon triple bond derived by the removal of one hydrogenatom front a single carbon atom of a parent alkyne. Typical alkynylgroups include, but are not limited to, ethynyl; propynyls such asprop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butynyls such as but-1-yn-1-yl,but-3-yn-1-yl, etc.; and the like.

The term “alkylamine” refers to a group of the formula —NHR^(xa) and“dialkylamine” refers to a group of the formula —NR^(xa)R^(xa), whereeach R^(xa) is, independently of the others, an alkyl group. The term“alkylene” refers to an alkane, allure or alkyne group having twoterminal monovalent radical centers derived by the removal of onehydrogen atom from each of the two terminal carbon atoms. Typicalalkylene groups include, but are not limited to, methylene; andsaturated or unsaturated ethylene; propylene; butylene; and the like.The term “lower alkylene” refers to alkylene groups with 1 to 6 carbons.

The term “heteroalkylene” refers to a divalent alkylene having one ormore —CH₂— groups replaced with a thio, oxy, or —NR^(x3)— where R^(x3)is selected from hydrogen, lower alkyl and lower heteroalkyl. Theheteroalkylene can be linear, branched, cyclic, bicyclic, or acombination thereof and can include up to 10 carbon atoms and up to 4heteroatoms. The term “lower heteroalkylene” refers to alkylene groupswith 1 to 4 carbon atoms and 1 to 3 heteroatoms.

The term “aryl” means an aromatic carbocyclyl containing from 6 to 14carbon ring atoms. An aryl may be monocyclic or poly cyclic (i.e., maycontain more than one ring). In the case of polycyclic aromatic rings,only one ring the polycyclic system is required to be aromatic while theremaining ring(s) may be saturated, partially saturated or unsaturated.Examples of aryls include phenyl, naphthalenyl, indenyl, indanyl, andtetrahydronaphthyl.

The term “arylene” refers to an aryl group having two monovalent radicalcenters derived by the removal of one hydrogen atom from each of the tworing carbons. An exemplary arylene group is a phenylene.

An alkyl group may be substituted by a “carbonyl” which means that twohydrogen atoms from a single alkanylene carbon atom are removed andreplaced with a double bond to an oxygen atom.

The prefix “halo” indicates that the substituent which includes theprefix is substituted with one or more independently selected halogenradicals. For example, haloalkyl means an alkyl substituent in which atleast one hydrogen radical is replaced with a halogen radical. Typicalhalogen radicals include chloro, fluoro, bromo and iodo. Examples ofhaloalkyls include chloromethyl, 1-bromoethyl, fluoromethyl,difluoromethyl, trifluoromethyl, and 1,1,1-trifluoroethyl. It should berecognized that if a substituent is substituted by more than one halogenradical, those halogen radicals may be identical or different (unlessotherwise stated).

The term “haloalkoxy” refers to a group of the formula OR^(c), whereR^(c) is a haloalkyl.

The terms “heteroalkyl,” “heteroalkanyl,” “heteroalkenyl,”“heteroalkynyl,” and “heteroalkylene” refer to alkyl, alkenyl, alkenyl,alkynyl, and alkylene groups, respectively, in which one or more of thecarbon atoms, e.g., 1, 2 or 3 carbon atoms, are each independentlyreplaced with the same or different heteroatoms or heteroatomic groups.Typical heteroatoms and/or heteroatomic groups which can replace thecarbon atoms include, but are not limited to, —O—, —S—, —S—O—, —NR^(c)—,—PH, —S(O)—, —S(O)₂—, —S(O)NR^(c)—, —S(O)₂NR^(c)—, and the like,including combinations thereof, where each RC is independently hydrogenor C₁-C₆ alkyl. The term “lower heteroalkyl” refers to between 1 and 4carbon atoms and between 1 and 3 heteroatoms.

The terms “cycloalkyl” and “heterocyclyl” refer to cyclic versions of“alkyl” and “heteroalkyl” groups, respectively. For heterocyclyl groups,a heteroatom can occupy the position that is attached to the remainderof the molecule. A cycloalkyl or heterocyclyl ring may be a single-ring(monocyclic) or have two or more rings (bicyclic or polycyclic).

Monocyclic cycloalkyl and heterocyclyl groups will typically containsfrom 3 to 7 ring atoms, more typically from 3 to 6 ring atoms, and evenmore typically 5 to 6 ring atoms. Examples of cycloalkyl groups include,but are not limited to, cyclopropyl; cyclobutyls such as cyclobutanyland cyclobutenyl; cyclopentyls such as cyclopentanyl and cyclopentenyl;cyclohexyls such as cyclohexanyl and cyclohexenyl; and the like.Examples of monocyclic heterocyclyls include, but are not limited to,oxetane, furanyl, dihydrofuranyl, tetrahydrofuranyl, tetrahydropyranyl,thiophenyl (thiofuranyl), dihydrothiophenyl, tetrahydrothiophenyl,pyrrolyl, pyrrolinyl, pyrrolidinyl, imidazolyl, imidazolinyl, pyrazolyl,pyrazolinyl, pyrazolidinyl, triazolyl, tetrazolyl, axazolyl,oxazolidinyl, isoxazolidinyl, isoxazolyl, thiazolyl, isothiazolyl,thiazolinyl, isothiazolinyl, thiazolidinyl, isothiazolidinyl,thiadiazolyl, oxadiazolyl (including 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl (furazanyl), or 1,3,4-oxadiazolyl),oxatriazolyl (including 1,2,3,4-oxatriazolyl or 1,2,3,5-oxatriazolyl),dioxazolyl (including 1,2,3-dioxazolyl, 1,2,4-dioxazolyl,1,3,2-dioxazolyl, or 1,3,4-diaxazolyl), 1,4 dioxanylydioxothionaorpholinyl, oxathiazolyl, oxathiolyl, oxathiolanyl, pyrranyl,dihydropyranyl, thiopyranyl, tetrahydrothiopyranyl, pyridinyl (azinyl),piperidinyl, diazinyl (including pyridazinyl (1,2-diazinyl), pyrimidinyl(1,3-diazinyl), or pyrazinyl (1,4-diazinyl)), piperazinyl, triazinyl(including 1,3,5-triazinyl, 1,2,4-triazinyl, and 1,2,3-triazinyl))),oxazinyl (including 1,2-oxazinyl, 1,3-oxazinyl, or 1,4-oxazinyl)),oxathiazinyl (including 1,2,3-oxathiazinyl, 1,2,4-oxathiazinyl,1,2,5-oxathiazinyl, or 1,2,6-oxathiazinyl)), oxadiazinyl (including1,2,3-oxadiazinyl, 1,2,4-oxadiazinyl, 1,4,2-oxadiazinyl, or1,3,5-oxadiazinyl)), morpholinyl, azepinyl, oxepinyl, thiepinyl,diazepinyl, pyridonyl (including pyrid-2(1H)-onyl and pyrid-4(1H)-onyl),furan-2(5H)-onyl, pyrimidonyl (including pyramid-2(1H)-onyl andpyramid-4(3H)-onyl), oxazol-2(3H)-onyl, 1H-imidazol-2(3H)-onyl,pyridazin-3(2H)-onyl, and pyrazin-2(1H)-onyl.

Polycyclic cycloalkyl and heterocyclyl groups contain more than onering, and bicyclic cycloalkyl and heterocyclyl groups contain two rings.The rings may be in a bridged, fused or spiro orientation. Polycycliccycloalkyl and heterocyclyl groups may include combinations of bridged,fused and/or spiro rings. In a spirocyclic cycloalkyl or heterocyclyl,one atom is common to two different rings. An example of aspirocycloalkyl is spiro[4.5]decane and an example of aspiroheterocyclyls is a spiropyrazoline.

In a bridged cycloalkyl or heterocyclyl, the rings share at least twocommon non-adjacent atoms. Examples of bridged cycloalkyls include, butare not limited to, adamantyl and norbornanyl rings. Examples of bridgedheterocyclyls include, but are not limited to,2-oxatricyclo[3.3.1.1^(3,7)]decanyl.

In a fused-ring cycloalkyl or heterocyclyl, two or more rings are fusedtogether, such that two rings share one common bond. Examples offused-ring cycloalkyls include decalin, naphthylene, tetralin, andanthracene. Examples of fused-ring heterocyclyls containing two or threerings include imidazopyrazinyl (including imidazo[1,2-a]pyrazinyl),imidazopyridinyl (including imidazo[1,2-a]pyridinyl), imidazopyridazinyl(including imidazo[1,2-b]pyridazinyl), thiazolopyridinyl (includingthiazolo[5,4-c]pyridinyl, thiazolo[5,4-b]pyridinyl,thiazolo[4,5-b]pyridinyl, and thiazolo[4,5-c]pyridinyl), indolzinyl,pyranopyrrolyl, 4H-quinolizinyl, purinyl, naphthyridinyl,pyridopyridinyl (including pyrido[3,4-h]pyridinyl,pyrido[3,2-b]-pyridinyl, or pyrido[4,3-b]pyridinyl), and pteridinyl.Other examples of fused-ring heterocyclyls include benzo-fusedheterocyclyls, such as dihydrochromenyl, tetrahydroisoquinolinyl,indolyl, isoindolyl (isobenzazolyl, pseudoisoindolyl), indoleninyl(pseudoindolyl), isoindazolyl (henzpyrazolyl), henzazinyl (includingquinolinyl (1-benzazinyl) or isoquinolinyl (2-benzazinyl)),phthalazinyl, quinoxalinyl, quinazolinyl, benzodiazinyl (includingcinnolinyl (1,2-bcnzodiazinyl) or quinazolinyl (1,3-benzodiazinyl)),benzopyranyl (including chromanyl or isochromanyl), benzoxazinyl(including 1,3,2-benzoxazinyl, 1,4,2-benzoxazinyl, 2,3,1-benzoxazinyl,or 3,1,4-benzoxazinyl), benzo[d]thiazolyl, and benzisoxazinyl (including1,2-benzisoxazinyl or 1,4-benzisoxazinyl).

The term “cycloalkylene” refers to a cycloalkyl group having twomonovalent radical centers derived by the removal of one hydrogen atomfrom each of two ring carbons. Exemplary cycloalkylene groups include:

The term “heteroaryl” refers to an aromatic heterocyclyl containing from5 to 14 ring atoms. A heteroaryl may be a single ring or 2 or 3 fusedrings. Examples of heteroaryls include 6-membered rings such as pyridyl,pyrazyl, pyrimidinyl, pyridazinyl, and 1,3,5-, 1,2,4- or1,2,3-triazinyl; 5-membered ring substituents such as triazolyl,pyrrolyl, imidazyl, furanyl, thiophenyl, pyrazolyl, oxazolyl,isoxazolyl, thiazolyl, 1,2,3-, 1,2,4-, 12,5-, or 1,3,4-oxadiazolyl andisothiazolyl 6/5-membered fused ring substituents such asimidazopyrazinyl (including imidazo[1,2-a]pyrazinyl)imidazopyridinyl(including imidazo[1,2-a]pyridinyl), imidazopyridazinyl (includingimidazo[1,2-b]pyridazinyl), thiazolopyridinyl (includingthiazolo[5,4-c]pyridinyl, thiazolo[5,4-b]pyridinyl,thiazolo[4,5-b]pyridinyl, and thiazolo[4,5-c]pyridinyl),benzo[d]thiazolyl, benzothiofuranyl, benzisoxazolyl, benzoxazolyl,purinyl, and anthranilyl; and 6/6-membered fused rings such asbenzopyranyl, quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl, andbenzoxazinyl. Heteroaryls may also be heterocycles having aromatic (4N+2pi electron) resonance contributors such as pyridonyl (includingpyrid-2(1H)-onyl and pyrid-4(1H)-onyl), pyrimidonyl (includingpyramid-2(1H)-onyl and pyramid-4(3H)-onyl), pyridazin-3(2H)-onyl andpyrazin-2(1H)-onyl.

The term “sulfonate” as used herein means a salt or ester of a sulfonicacid.

The term “methyl sulfonate” as used herein means a methyl ester of asulfonic acid group.

The term “carboxy late” as used herein means a salt or ester of acarboxylic acid.

The term “polyol”, as used herein, means a group containing more thantwo hydroxyl groups independently or as a portion of a monomer unit.Polyols include, but are not limited to, reduced C₂-C₆ carbohydrates,ethylene glycol, and glycerin.

The term “sugar” when used in context of “G¹” includes O-glycoside,N-glycoside, S-glycoside and C-glycoside (C-glycoslyl) carbohydratederivatives of the monosaccharide and disaccharide classes and mayoriginate from naturally-occurring sources or may be synthetic inorigin. For example “sugar” when used in context of “G¹” includesderivatives such as but not limited to those derived from glucuronicacid, galacturonic acid, galactose, and glucose among others. Suitablesugar substitutions include but are not limited to hydroxyl, amine,carboxylic acid, sulfonic acid, phosphonic acid, esters, and ethers.

The term “NHS ester” means the N-hydroxysuccinimide ester derivative ofa carboxylic acid.

The term “amine” includes primary, secondary and tertiary aliphaticamines, including cyclic versions.

The term salt when used in context of “or salt thereof” include saltscommonly used to form alkali metal salts and to form addition salts offree acids or free bases. In general, these salts typically may beprepared by conventional means by reacting, for example, the appropriateacid or base with a compound of the invention Where a salt is intendedto be administered to a patient (as opposed to, for example, being inuse in an in vitro context), the salt preferably is pharmaceuticallyacceptable and/or physiologically compatible. The term “pharmaceuticallyacceptable” is used adjectivally in this patent application to mean thatthe modified noun is appropriate for use as a pharmaceutical product oras a part of a pharmaceutical product. The term “pharmaceuticallyacceptable salt” includes salts commonly used to form alkali metal saltsand to form addition salts of free acids or free bases. In general,these salts typically may be prepared by conventional means by reacting,for example, the appropriate acid or base with a compound of theinvention.

Various aspects of the invention are described in further detail in thefollowing subsections.

II. Anti-B7-H3 Antibodies

One aspect of the invention provides anti-B7-H3 antibodies, or antigenbinding portions thereof. In one embodiment, the present inventionprovides chimeric anti-B7-H3 antibodies, or antigen binding portionsthereof. In yet another embodiment, the present invention provideshumanized anti-B7-H3 antibodies, or antigen binding portions thereof. Inanother aspect, the invention features antibody drug conjugates (ADCs)comprising an anti-B7-H3 antibody described herein and at least onedrug(s), such as, but not limited to, a Bcl-xL inhibitor or anauristatin. The antibodies or ADCs of the invention have characteristicsincluding, but not limited to, binding to wild-type human B7-H3 invitro, binding to wild-type human B7-H3 on tumor cells expressing B7-H3,and decreasing or inhibiting xenograft tumor growth in a mouse model.

One aspect of the invention features an anti-human B7-H3 (anti-hB7-H3)Antibody Drug Conjugate (ADC) comprising an anti-hB7-H3 antibodyconjugated to a drug via a linker, wherein the drug is a Bcl-xLinhibitor. Exemplary anti-B7-H3 antibodies (and sequences thereof) thatcan be used in the ADCs are described herein.

The anti-B7-H3 antibodies described herein provide the ADCs of theinvention with the ability to bind to B7-H3 such that the cytotoxicBcl-xl, drug attached to the antibody may be delivered to theB7-H3-expressing cell, particularly a B7-H3 expressing cancer cell.

While the term “antibody” is used throughout, it should be noted thatantibody fragments (i.e., antigen-binding portions of an anti-B7-H3antibody) are also included in the invention and may be included in theembodiments (methods and compositions) described throughout. Forexample, an anti-B7-H3 antibody fragment may be conjugated to the Bcl-xLinhibitors described herein. Thus, it is within the scope of theinvention that in certain embodiments, antibody fragments of theanti-B7-H3antibodies described herein are conjugated to Bcl-xLinhibitors (including those described below in Section III.A) vialinkers (including those described below in Section IIIA). In certainembodiments, the anti-B7-H3 antibody binding portion is a Fab, a Fab′, aF(ab′)2, a Fv, a disulfide linked Fv, an scFv, a single domain antibody,or a diabody.

II.A. Anti-B7-H3 Chimeric Antibodies

A chimeric antibody is a molecule in which different portions of theantibody are derived from different animal species, such as antibodieshaving a variable region derived from a murine monoclonal antibody and ahuman immunoglobulin constant region. Methods for producing chimericantibodies are known in the art. See e.g., Morrison, Science 229:1202(1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J.Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and4,816,397, which are incorporated herein by reference in theirentireties. In addition, techniques developed for the production of“chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad Sci.81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al.,1985, Nature 314:452-454, each of which are incorporated herein byreference in their entireties) by splicing genes from a mouse antibodymolecule of appropriate antigen specificity together with genes from ahuman antibody molecule of appropriate biological activity can be used.

As described in Example 3, eighteen anti-B7-H3 murine antibodies wereidentified having high specific binding activity against human andcynomolgus B7-H3. Chimeric antibodies, in the context of a humanimmunoglobulin constant region, were generated from these eighteenantibodies.

Thus, in one aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence set forth in SEQ IDNOs: 1, 9, 16, 24, 32, 40, 48, 56, 64, 72, 80, 87, 95, 101, or 108;and/or a light chain variable region including an amino acid sequenceset forth in SEQ ID NOs: 5, 13, 20, 28, 36, 44, 52, 60, 68, 76, 84, 91,98, 105, or 112.

In another aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 1, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 5.

In another aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable domain region including (a) a CDR1 having an amino acidsequence as set forth in SEQ ID NO: 2; (b) a CDR2 having an amino acidsequence as set forth in SEQ ID NO: 3; and (c) a CDR3 having an aminoacid sequence as set forth in SEQ ID NO: 4; and a light chain variableregion including (a) a CDR1 having an amino acid sequence as set forthin SEQ ID NO: 6; (b) a CDR2 having an amino acid sequence as set forthin SEQ ID NO: 7; and (c) a CDR3 having an amino acid sequence as setforth in SEQ ID NO: 8.

In another aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 9, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 13.

In another aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable domain region including (a) a CDR1 having an amino acidsequence as set forth in SEQ ID NO: 10; (b) a CDR2 having an amino acidsequence as set forth in SEQ ID NO: 11; and (c) a CDR3 having an aminoacid sequence as set forth in SEQ ID NO: 12; and a light chain variableregion including (a) a CDR1 having an amino acid sequence as set forthin SEQ ID NO: 14 (b) a CDR2 having an amino acid sequence as set forthin SEQ ID NO: 7;

and (c) a CDR3 having an amino acid sequence as set forth in SEQ ID NO:1.5.

In another aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 16, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 20.

In another aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable domain region including (a) a CDR1 having an amino acidsequence as set forth in SEQ ID NO: 17; (b) a CDR2 having an amino acidsequence as set forth in SEQ ID NO: 18; and (c) a CDR3 having an aminoacid sequence as set forth in SEQ ID NO: 19; and a light chain variableregion including (a) a CDR1 having an amino acid sequence as set forthin SEQ ID NO: 21; (b) a CDR2 having an amino acid sequence as set forthin SEQ ID NO: 22; and (c) a CDR3 having an amino acid sequence as setforth in SEQ ID NO: 23.

In another aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 24, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 28.

In another aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable domain region including (a) a CDR1 having an amino acidsequence as set forth in SEQ ID NO: 25; (b) a CDR2 having an amino acidsequence as set forth in SEQ ID NO: 26; and (c) a CDR3 having an aminoacid sequence as set forth in SEQ ID NO: 27; and a light chain variableregion including (a) a CDR1 having an amino acid sequence as set forthin SEQ ID NO: 29; (b) a CDR2 having an amino acid sequence as set forthin SEQ ID NO: 30; and (c) a CDR3 having an amino acid sequence as setforth in SEQ ID NO: 31.

In another aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 32, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 36.

In another aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable domain region including (a) a CDR1 having an amino acidsequence as set forth in SEQ ID NO: 33; (b) a CDR2 having an amino acidsequence as set forth in SEQ ID NO: 34; and (c) a CDR3 having an aminoacid sequence as set forth in SEQ ID NO: 35; and a light chain variableregion including (a) a CDR1 having an amino acid sequence as set forthin SEQ ID NO: 37; (b) a CDR2 having an amino acid sequence as set forthin SEQ ID NO: 38; and (c) a CDR3 having an amino acid sequence as setforth in SEQ ID NO: 182, In another aspect, the present invention isdirected to an anti-B7-H3 antibody, or antigen-binding portion thereof,having a heavy chain variable region including an amino acid sequence asset forth in SEQ ID NO: 40, and a light chain variable region includingan amino acid sequence set forth in SEQ ID NO: 44.

In another aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable domain region including (a) a CDR1 having an amino acidsequence as set forth in SEQ ID NO: 41; (b) a CDR2 having an amino acidsequence as set forth in SEQ ID NO: 42; and (c) a CDR3 having an aminoacid sequence as set forth in SEQ ID NO: 43; and a light chain variableregion including (a) a CDR1 having an amino acid sequence as set forthin SEQ ID NO: 45; (b) a CDR2 having an amino acid sequence as set forthin SEQ ID NO: 46; and (c) a CDR3 having an amino acid sequence as setforth in SEQ ID NO: 47.

In another aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 48, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 52.

In another aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable domain region including (a) a CDR1, having an amino acidsequence as set forth in SEQ ID NO: 49; (b) a CDR2 having an amino acidsequence as set forth in SEQ ID NO: 50; and (c) a CDR3 having an aminoacid sequence as set forth in SEQ ID NO: 51, and a light chain variableregion including (a) a CDR1 having an amino acid sequence as set forthin SEQ ID NO: 53; (b) a CDR2 having an amino acid sequence as set forthin SEQ ID NO: 54; and (c) a CDR3 having an amino acid sequence as setforth in SEQ ID NO: 55.

In another aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 56, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 60.

In another aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable domain region including (a) a CDR1 having an amino acidsequence as set forth in SEQ ID NO: 57; (b) a CDR2 having an amino acidsequence as set forth in SEQ ID NO: 58; and (c) a CDR3 having an aminoacid sequence as set forth in SEQ ID NO: 59; and a light chain variableregion including (a) a CDR1 having an amino acid sequence as set forthin SEQ ID NO: 61; (b) a CDR2 having an amino acid sequence as set forthin SEQ ID NO: 62; and (c) a CDR3 having an amino acid sequence as setforth in SEQ ID NO: 63.

In another aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 64, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 68.

In another aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable domain region including (a) a CDR1 having an amino acidsequence as set forth in SEQ ID NO: 65; (b) a CDR2 having an amino acidsequence as set forth in SEQ ID NO: 66; and (c) a CDR3 having an aminoacid sequence as set forth in SEQ ID NO: 67; and a light chain variableregion including (a) a CDR1 having an amino acid sequence as set forthin SEQ ID NO: 69; (b) a CDR2 having an amino acid sequence as set forthin SEQ ID NO: 70; and (c) a CDR3 having an amino acid sequence as setforth in SEQ ID NO: 71.

In another aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 72, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 76.

In another aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable domain region including (a) a CDR1 having an amino acidsequence as set forth in SEQ ID NO: 73; (b) a CDR2 having an amino acidsequence as set forth in SEQ ID NO: 74, and (c) a CDR3 having an aminoacid sequence as set forth in SEQ ID NO: 75; and a light chain variableregion including (a) a CDR1 having an amino acid sequence as set forthin SEQ ID NO: 77; (b) a CDR2 having an amino acid sequence as set forthin SEQ ID NO: 78; and (c) a CDR3 having an amino acid sequence as setforth in SEQ ID NO: 79.

In another aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 80, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 84.

In another aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable domain region including (a) a CDR1 having an amino acidsequence as set forth in SEQ ID NO: 81; (b) a CDR2 having an amino acidsequence as set forth in SEQ ID NO: 82; and (c) a CDR3 having an aminoacid sequence as set forth in SEQ ID NO: 83; and a light chain variableregion including (a) a CDR1 having an amino acid sequence as set forthin SEQ ID NO: 85; (b) a CDR2 having an amino acid sequence as set forthin SEQ ID NO: 7; and (c) a CDR3 having an amino acid sequence as setforth in SEQ ID NO: 86.

In another aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 87, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 91.

In another aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable domain region including (a) a CDR1 having an amino acidsequence as set forth in SEQ ID NO: 88; (b) a CDR2 having an amino acidsequence as set forth in SEQ ID NO: 89; and (c) a CDR3 having an aminoacid sequence as set forth in SEQ ID NO: 90; and a light chain variableregion including (a) a CDR1 having an amino acid sequence as set forthin SEQ ID NO: 92; (b) a CDR2 having an amino acid sequence as set forthin SEQ ID NO: 93; and (c) a CDR3 having an amino acid sequence as setforth in SEQ ID NO: 94.

In another aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 95, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 98.

In another aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable domain region including (a) a CDR1 having an amino acidsequence as set forth in SEQ ID NO: 49; (b) a CDR2 having an amino acidsequence as set forth in SEQ ID NO: 96; and (c) a CDR3 having an aminoacid sequence as set forth in SEQ ID NO: 97; and a light chain variableregion including (a) a CDR1 having an amino acid sequence as set forthin SEQ ID NO: 99; (b) a CDR2 having an amino acid sequence as set forthin SEQ ID NO: 93; and (c) a CDR3 having an amino acid sequence as setforth in SEQ ID NO: 100.

In another aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 101, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 105.

In another aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable domain region including (a) a CDR1 having an amino acidsequence as set forth in SEQ ID NO: 102; (b) a CDR2 having an amino acidsequence as set forth in SEQ ID NO: 103; and (c) a CDR3 having an aminoacid sequence as set forth in SEQ NO: 104; and a light chain variableregion including (a) a CDR1 having an amino acid sequence as set forthin SEQ ID NO: 106; (b) a CDR2 having an amino acid sequence as set forthin SEQ ID NO: 46; and (c) a CDR3 having an amino acid sequence as setforth in SEQ ID NO: 107.

In another aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 108, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 112.

In another aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable domain region including (a) a CDR1 having an amino acidsequence as set forth in SEQ ID NO: 109; (b) a CDR2 having an amino acidsequence as set forth in SEQ ID NO: 110; and (c) a CDR3 having an aminoacid sequence as set forth in SEQ ID NO: 111; and a light chain variableregion including (a) a CDR1 having an amino acid sequence as set forthin SEQ ID NO: 113; (b) a CDR2 having an amino acid sequence as set forthin SEQ ID NO: 114; and (c) a CDR3 having an amino acid sequence as setforth in SEQ ID NO: 115.

II.B. Humanized Anti-B7-H3 Antibodies

The chimeric antibodies disclosed herein may be used in the productionof humanized anti-B7-H3 antibodies. For example, following thegeneration and characterization of chimeric anti-B7-H3 antibodieschAb1-chAb18, antibodies chAb3; chAh13, and chAb18 were selected forhumanization. Specifically, six different humanized antibodies werecreated based on chAb3 (referred to herein as huAb3v1, huAb3v2, huAb3v3,huAb3v4, huAb3v5, and huAb3v6 (see Examples 12 and 13), nine differenthumanized antibodies were created based on chAb13 (referred to herein ashuAb13v1, huAb13v2, huAb13v3, huAb13v4, huAb13v5, huAb13v6, huAb1.3v7,huAb13v8; huAb13v9), and ten different humanized antibodies were createdbased on chAb18 (referred to herein as huAb18v1, huAb18v2, huAb18v3,huAb18v4, huAb18v5, huAb18v6, huAb18v7, huAb18v8, huAb18v9, andhuAb18v10 (see Examples 9 and 10)). Tables 8, 12, 16, 18, and 19 providethe amino acid sequences of CDR, VH and VL regions of humanized chAb3,chAb13, and chAb1.8, respectively.

Generally, humanized antibodies are antibody molecules from non-humanspecies antibody that binds the desired antigen having one or morecomplementarity determining regions (CDRs) from the non-human speciesand framework regions from a human immunoglobulin molecule. Known humanIg sequences are disclosed, e.g.,www.ncbi.nlm.nih.gov/entrez-/query.fcgi; www.atcc.org/phage/hdb.html;www.sciquest.com/; www.abcam.com/;www.antibodyresource.com/onlinecomp.html;www.public.iastate.edu/.about.pedro/research_tools.html;www.mgen.uni-heidelberg.de/SD/IT/IT.html;www.whfreeman.com/immunology/CH-05/kuby05.htm;www.library.thinkquest.org/12429/Immune/Antibody.html;www.hhmi.org/grants/lectures/1996/vlab/;www.path.cam.ac.uk/.about.mrc7/m-ikeimages.html;www.antibodyresource.com/;mcb.harvard.edu/BioLinks/Immuno-logy.html.www.immunologylink.com/;pathbox.wustl.edu/.about.hcenter/index.-html;www.biotech.ufl.edu/.about.hcl/; www.pebio.com/pa/340913/340913.html-;www.nal.usda.gov/awic/pubs/antibody/;www.m.ehime-u.acjp/.about.yasuhito-/Elisa.html;www.biodesign.com/table.asp; www.icnet.uk/axp/facs/davies/lin-ks.html;www.biotech.ufl.edu/.about.fcclprotocol.html;www.isac-net.org/sites_geo.html;aximtl.imt.uni-marburg.de/.about.rek/AEP-Start.html;baserv.uci.kun.nl/.about.jraats/linksl.html;www.recab.uni-hd.de/immuno.bme.nwu.edu/;www.mrc-cpe.cam.ac.uk/imt-doc/pu-blic/INTRO.html;www.ibt.unam.mx/vir/V_mice.html; imgt.cnusc.fr:8104/;www.biochem.ucl.ac.uk/.about.martin/abs/index.html;antibody.bath.ac.uk/; abgen.cvm.tamu.edu/lab/wwwabgen.html;www.unizh.ch/.about.honegger/AHOsem-inar/Slide01.html;www.cryst.bbk.ac.uk/.about.ubcg07s/;www.nimr.mrc.ac.uk/CC/ccaewg/ccaewg.htm;www.path.cam.ac.uk/.about.mrc7/h-umanisation/TAHHP.html;www.ibt.unam.mx/vir/structure/stat_aim.html;www.biosci.missouri.edu/smithgp/index.html;www.cryst.bioc.cam.ac.uk/.abo-ut.fmolina/Web-pages/Pept/spottech.html;www.jerini.de/fr roducts.htm; www.patents.ibm.com/ibm.html.Kabat et al.,Sequences of Proteins of Immunological Interest, U.S. Dept. Health(1983), each entirely incorporated herein by reference. Such importedsequences can be used to reduce immunogenicity or reduce, enhance ormodify binding, affinity, on-rate, off-rate, avidity, specificity,half-life, or any other suitable characteristic, as known in the art.

Framework residues in the human framework regions may be substitutedwith the corresponding residue from the CDR donor antibody to alter,preferably improve, antigen binding. These framework substitutions areidentified by methods well known in the art, e.g., by modeling of theinteractions of the CDR and framework residues to identify frameworkresidues important for antigen binding and sequence comparison toidentify unusual framework residues at particular positions. (See, e.g.,Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323(1988), which are incorporated herein by reference in their entireties.)Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.Antibodies can be humanized using a variety of techniques known in theart, such as but not limited to those described in Jones et al., Nature321:522 (1986); Verhoeyen et al., Science 239:1534 (1988)), Sims et al.,J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901(1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285 (1992);Presta et al., J. Immunol. 151:2623 (1993), Padlan, Molecular Immunology28(4/5):489-498 (1991); Studnicka et al., Protein Engineering7(6):805-814 (1994); Roguska. et al.,PNAS 91:969-973 (1994); PCTpublication WO 91/09967, PCT/: US98/16280, US96/18978, US91/09630,US91/05939, US94/01234, GB89/01334, GB91/01134, GB92/01755; WO90/14443,WO90/14424, WO90/14430, EP 229246, EP 592,106; EP 519,596, EP 239,400,U.S. Pat. Nos. 5,565,332, 5,723,323, 5,976,862, 5,824,514, 5,817,483,5,814,476, 5,763,192, 5,723,323, 5,766886, 5,714,352, 6,204,023,6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539; 4,816,567, eachentirely incorporated herein by reference, included references citedtherein.

Humanized Anti-B7-H3 Antibodies Derived from chAb3

Six humanized antibodies based on chAb3 were created. The sequences ofeach are as follows:

A) huAb3v1 (VH amino acid sequence set forth in SEQ ID NO: 125 and VHCDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 10,11, and 12, respectively; and VL amino acid sequence set forth in SEQ IDNO: 128 and VL CDR1, CDR2, and CDR3 amino acid sequences set forth inSEQ ID NOs: 14, 7, and 15, respectively);

B) huAb3v2 (VH amino acid sequence set forth in SEQ ID NO: 127 and VHCDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 10,11, and 12, respectively; and VL amino acid sequence set forth in SEQ IDNO: 128 and VL CDR1, CDR2, and CDR3 amino acid sequences set forth inSEQ ID NOs: 14, 7, and 15, respectively);

C) huAb3v3 (VH amino acid sequence set forth in SEQ ID NO: 126 and VHCDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 10,11, and 12, respectively; and VL amino acid sequence set forth in SEQ IDNO: 129 and VL CDR1, CDR2, and CDR3 amino acid sequences set forth inSEQ ID NOs: 14, 7, and 15, respectively);

D) huAb3v4 (VH amino acid sequence set forth in SEQ ID NO: 125 and VHCDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 10,11, and 12, respectively; and VL amino acid sequence set forth in SEQ IDNO: 130 and VL CDR1, CDR2, and CDR3 amino acid sequences set forth inSEQ ID NOs: 14, 7, and 15, respectively);

E) huAb3v5 (VH amino acid sequence set forth in SEQ ID NO: 127 and VHCDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 10,11, and 12, respectively; and VL amino acid sequence set forth in SEQ IDNO: 130 and VL CDR1, CDR2, and CDR3 amino acid sequences set forth inSEQ ID NOs: 14, 7, and 15, respectively); and

F) huAb3v6 (VH amino acid sequence set forth in SEQ ID NO: 126 and VHCDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 10,11, and 12, respectively; and VL amino acid sequence set forth in SEQ IDNO: 130 and VL CDR1, CDR2, and CDR3 amino acid sequences set forth inSEQ ID NOs: 14, 7, and 15, respectively).

Of the six humanized versions of chAb3, huAb3v2 was selected for furthermodified in order to remove potential deamidation or isomerization sitesin the light chain CDR1 or in the heavy chain CDR2. Nine variants of thehumanized antibody huAb3v2 were generated, and are referred to herein ashuAb3v2.1, huAb3v2.2, huAb3v2.3, huAb3v2.4, huAb3v2.5, huAb3v16,huAb3v2.7, huAb3v2.8, and huAb3v2.9 (CDR and variable domain sequencesare provided in Table 16). The nine variants of the huAb3v2 antibodyinclude the following:

A) huAb3v2.1 (VH amino acid sequence set forth in SEQ ID NO: 131 and VHCDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 10,132, and 12, respectively; and VL amino acid sequence set forth in SEQID NO: 133 and VL CDR1, CDR2, and CDR3 amino acid sequences set forth inSEQ ID NOs: 134, 7, and 15, respectively);

B) huAb3v2.2 (VH amino acid sequence set forth in SEQ ID NO: 131 and VHCDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 10,132, and 12, respectively; and VL amino acid sequence set forth in SEQID NO: 135 and VL CDR1, CDR2, and CDR3 amino acid sequences set forth inSEQ ID NOs: 136, 7, and 15, respectively);

C) huAb3v2.3 (VH amino acid sequence set forth in SEQ ID NO: 131 and VHCDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 10,132, and 12, respectively; and VL amino acid sequence set forth in SEQID NO: 137 and VL CDR1, CDR2, and CDR3 amino acid sequences set forth inSEQ ID NOs: 138, 7, and 15, respectively);

D) huAb3v2.4 (VH amino acid sequence set forth in SEQ ID NO: 139 and VHCDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 10,140, and 12, respectively; and VL amino acid sequence set forth in SEQID NO: 133 and VL CDR1, CDR2, and CDR3 amino acid sequences set forth inSEQ ID NOs: 134, 7, and 15, respectively);

E) huAb3v2.5 (VH amino acid sequence set forth in SEQ ID NO: 139 and VHCDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 10,140, and 12, respectively; and VL amino acid sequence set forth in SEQID NO: 135 and VL CDR1, CDR2, and CDR3 amino acid sequences set forth inSEQ ID NOs: 136, 7, and 15, respectively);

F) huAb3v2.6 (VH amino acid sequence set forth in SEQ ID NO: 139 and VHCDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 10,140, and 12, respectively; and VL amino acid sequence set forth in SEQID NO: 137 and VL CDR1, CDR2, and CDR3 amino acid sequences set forth inSEQ ID NOs: 138, 7, and 15, respectively);

G) huAb3v2.7 (VH amino acid sequence set forth in SEQ ID NO: 141 and VHCDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 10,142, and 12, respectively; and VL amino acid sequence set forth in SEQID NO: 133 and VL CDR1, CDR2, and CDR3 amino acid sequences set forth inSEQ ID NOs: 134, 7, and 15, respectively);

H) huAb3v2.8 (VH amino acid sequence set forth in SEQ ID NO: 141 and VHCDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 10,142, and 12, respectively; and VL amino acid sequence set forth in SEQID NO: 135 and VL CDR1, CDR2, and CDR3 amino acid sequences set forth inSEQ ID NOs: 136, 7, and 15, respectively); and

I) huAb3v2.9 (VH amino acid sequence set forth in SEQ ID NO: 141 and VHCDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 10,142, and 12, respectively; and VL amino acid sequence set forth in SEQID NO: 137 and VL CDR1, CDR2, and CDR3 amino acid sequences set forth inSEQ ID NOs: 138, 7, and 15, respectively).

Thus, in one aspect, the present invention provides antibodiescomprising variable and/or CDR sequences from a humanized antibodyderived from chAb3. In one embodiment, the invention features anti-B7-H3antibodies which are derived from Ab3 have improved characteristics,e.g., improved binding affinity to isolated B7-H3 protein and improvedbinding to B7-H3 expressing cells, as described in the Examples below.Collectively these novel antibodies are referred to herein as “Ab3variant antibodies.” Generally, the Ab3 variant antibodies retain thesame epitope specificity as Ab3. In various embodiments, anti-B7-H3antibodies, or antigen binding fragments thereof, of the invention arecapable of modulating a biological function of B7-H3.

In one aspect, the present invention provides a humanized antibody, orantigen binding portion thereof, having a heavy chain variable regionincluding an amino acid sequence set forth in SEQ ID NOs: 125, 126, 127,131, 139, or 141; and/or a light chain variable region including anamino acid sequence set forth in SEQ ID NOs: 128, 129, 130, 133, 135, or137.

In another aspect, the present invention is directed to an anti-B7-H3antibody, or antigen binding portion thereof, of the invention comprisesa heavy chain variable region comprising a CDR1 domain comprising anamino acid sequence as set forth in SEQ ID NO: 10, a CDR2 domaincomprising an amino acid sequence as set forth in SEQ ID NO: 11, 132,140, or 142; and a CDR3 domain comprising an amino acid sequence as setforth in SEQ ID NO: 12; and a light chain variable region comprising aCDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO:14, 134, 136, or 138; a CDR2 domain comprising an amino acid sequence asset forth in SEQ ID NO: 7; and a CDR3 domain comprising an amino acidsequence as set forth in SEQ ID NO: 15.

In one aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 125, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 128.

In one aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 127, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 128.

In one aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 126, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 129.

In one aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 125, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 130.

In one aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 127, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 130.

In one aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 126, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 130.

In another aspect, the present invention is directed to a humanizedanti-B7-H3 antibody, or antigen-binding portion thereof, having a heavychain variable domain region including (a) a CDR1 having an amino acidsequence as set forth in SEQ ID NO: 10; (b) a CDR2 having an amino acidsequence as set forth in SEQ ID NO: 11; and (c) a CDR3 having an aminoacid sequence as set forth in SEQ ID NO: 12; and a light chain variableregion including (a) a CDR1, having an amino acid sequence as set forthin SEQ ID NO: 14; (b) a CDR2, having an amino acid sequence as set forthin SEQ ID NO: 7; and (c) a CDR3 having an amino acid sequence as setforth in SEQ ID NO: 15.

In one aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 131, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 133.

In another aspect, the present invention is directed to a humanizedanti-B7-H3 antibody, or antigen-binding portion thereof, having a heavychain variable domain region including (a) a CDR1 having an amino acidsequence as set forth in SEQ ID NO: 10; (b) a CDR2 having an amino acidsequence as set forth in SEQ ID NO: 132; and (c) a CDR3 having an aminoacid sequence as set forth in SEQ ID NO: 12; and a light chain variableregion including (a) a CDR1 having an amino acid sequence as set forthin SEQ ID NO: 134; (b) a CDR2 having an amino acid sequence as set forthin SEQ ID NO: 7; and (c) a CDR3 having an amino acid sequence as setforth in SEQ ID NO: 15.

In one aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 131, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 135.

In another aspect, the present invention is directed to a humanizedanti-B7-H3 antibody, or antigen-binding portion thereof, having a heavychain variable domain region including (a) a CDR1 having an amino acidsequence as set forth in SEQ ID NO: 10; (b) a CDR2 having an amino acidsequence as set forth in SEQ ID NO: 132; and (c) a CDR3 having an aminoacid sequence as set forth in SEQ ID NO: 12; and a light chain variableregion including (a) a CDR1 having an amino acid sequence as set forthin SEQ ID NO: 136; (b) a CDR2 having an amino acid sequence as set forthin SEQ ID NO: 7; and (c) a CDR3 having an amino acid sequence as setforth in SEQ ID NO: 15, In one aspect, the present invention is directedto an anti-B7-H3 antibody, or antigen-binding portion thereof, having aheavy chain variable region including an amino acid sequence as setforth in SEQ ID NO: 131, and a light chain variable region including anamino acid sequence set forth in SEQ ID NO: 137.

In another aspect, the present invention is directed to a humanizedanti-B7-H3 antibody, or antigen-binding portion thereof, having a heavychain variable domain region including (a) a CDR1 having an amino acidsequence as set forth in SEQ ID NO: 10; (b) a CDR2 having an amino Acidsequence as set forth in SEQ ID NO: 132; and (c) a CDR3 having an aminoacid sequence as set forth in SEQ ID NO: 12; and a light chain variableregion including (a) a CDR1 having an amino acid sequence as set forthin SEQ ID NO: 138; (b) a CDR2 having an amino acid sequence as set forthin SEQ ID NO: 7; and (c) a CDR3 having an amino acid sequence as setforth in SEQ ID NO: 15.

In one aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 139, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 133.

In another aspect, the present invention is directed to a humanizedanti-B7-H3 antibody, or antigen-binding portion thereof, having a heavychain variable domain region including (a) a CDR1 having an amino acidsequence as set forth in SEQ ID NO: 10; (b) a CDR2 having an amino acidsequence as set forth in SEQ ID NO: 140; and (c) a CDR3 having an aminoacid sequence as set forth in SEQ ID NO: 12; and a light chain variableregion including (a) a CDR1 having an amino acid sequence as set forthin SEQ ID NO: 134; (b) a CDR2 having an amino acid sequence as set forthin SEQ ID NO: 7; and (c) a CDR3 having an amino acid sequence as setforth in SEQ ID NO: 15.

In one aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 139, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 135.

In another aspect, the present invention is directed to a humanizedanti-B7-H3 antibody, or antigen-binding portion thereof, having a heavychain variable domain region including (a) a CDR1 having an amino acidsequence as set forth in SEQ ID NO: 10; (b) a CDR2 having an amino acidsequence as set forth in SEQ ID NO: 140; and (c) a CDR3 having an aminoacid sequence as set forth in SEQ ID NO: 12; and a light chain variableregion including (a) a CDR1 having an amino acid sequence as set forthin SEQ ID NO: 136; (b) a CDR2 having an amino acid sequence as set forthin SEQ ID NO: 7; and (c) a CDR3 having an amino acid sequence as setforth in SEQ ID NO: 15.

In another aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chaincomprising the amino acid sequence of SEQ ID NO: 170 and a light chaincomprising the amino acid sequence of SEQ ID NO: 171.

In one aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 139, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 137.

In another aspect, the present invention is directed to a humanizedanti-B7-H3 antibody, or antigen-binding portion thereof, having a heavychain variable domain region including (a) a CDR1 having an amino acidsequence as set forth in SEQ ID NO: 10; (b) a CDR2 having an amino Acidsequence as set forth in SEQ ID NO: 140; and (c) a CDR3 having an aminoacid sequence as set forth in SEQ ID NO: 12; and a light chain variableregion including (a) a CDR1 having an amino acid sequence as set forthin SEQ ID NO: 138; (h) a CDR2 having an amino acid sequence as set forthin SEQ ID NO: 7; and (c) a CDR3 having an amino acid sequence as setforth in SEQ ID NO: 15.

In another aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chaincomprising the amino acid sequence of SEQ ID NO: 172 and a light chaincomprising the amino acid sequence of SEQ ID NO: 173. In one aspect, thepresent invention is directed to an anti-B7-H3 antibody, orantigen-binding portion thereof, having a heavy chain variable regionincluding an amino acid sequence as set forth in SEQ ID NO: 141, and alight chain variable region including an amino acid sequence set forthin SEQ ID NO: 133.

In another aspect, the present invention is directed to a humanizedanti-B7-H3 antibody, or antigen-binding portion thereof, having a heavychain variable domain region including (a) a CDR1 having an amino acidsequence as set forth in SEQ ID NO: 10; (b) a CDR2 having an amino acidsequence as set forth in SEQ ID NO: 142; and (c) a CDR3 having an aminoacid sequence as set forth in SEQ ID NO: 12; and a light chain variableregion including (a) a CDR1 having an amino acid sequence as set forthin SEQ ID NO: 134; (b) a CDR2 having an amino acid sequence as set forthin SEQ ID NO: 7; and (c) a CDR3 having an amino acid sequence as setforth in SEQ ID NO: 15.

In one aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 141, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 135.

In another aspect, the present invention is directed to a humanizedanti-B7-H3 antibody, or antigen-binding portion thereof, having a heavychain variable domain region including (a) a CDR1 having an amino acidsequence as set forth in SEQ ID NO: 10; (b) a CDR2 having an amino acidsequence as set forth in SEQ ID NO: 142; and (c) a CDR3 having an aminoacid sequence as set forth in SEQ ID NO: 12; and a light chain variableregion including (a) a CDR1 having an amino acid sequence as set forthin SEQ ID NO: 136; (b) a CDR2 having an amino acid sequence as set forthin SEQ ID NO: 7; and (c) a CDR3 having an amino acid sequence as setforth in SEQ ID NO: 15.

In one aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 141, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 137.

In another aspect, the present invention is directed to a humanizedanti-B7-H3 antibody, or antigen-binding portion thereof, having a heavychain variable domain region including (a) a CDR1 having an amino acidsequence as set forth in SEQ ID NO: 10; (b) a CDR2 having an amino acidsequence as set forth in SEQ ID NO: 142; and (c) a CDR3 having an aminoacid sequence as set forth in SEQ ID NO: 12; and a light chain variableregion including (a) a CDR1 having an amino acid sequence as set forthin SEQ ID NO: 138; (b) a CDR2 having an amino acid sequence as set forthin SEQ ID NO: 7; and (c) a CDR3 having an amino acid sequence as setforth in SEQ ID NO: 15.

Humanized Anti-B7-H3 Antibodies Derived from chAb13

The nine different humanized antibodies created based on chAb13 includethe following: A) huAb13v1 (VH amino acid sequence set forth in SEQ IDNO: 147 and VH CDR1, CDR2, and CDR3 amino acid sequences set forth inSEQ ID NOs: 33, 34, and 35, respectively; and VL amino acid sequence setforth in SEQ ID NO: 144 and VL CDR1, CDR2, and CDR3 amino acid sequencesset forth in SEQ ID NOs: 37, 38, and 39, respectively);

B) huAb13v2 (VH amino acid sequence set forth in SEQ ID NO: 146 and VHCDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 33,34, and 35, respectively; and VL amino acid sequence set forth in SEQ IDNO: 143 and VL CDR1, CDR2, and CDR3 amino acid sequences set forth inSEQ ID NOs: 37, 38, and 39, respectively);

C) huAb13v3 (VH amino acid sequence set forth in SEQ ID NO: 146 and VHCDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 33,34, and 35, respectively; and VL amino acid sequence set forth in SEQ IDNO: 144 and VL CDR1, CDR2, and CDR3 amino acid sequences set forth inSEQ ID NOs: 37, 38, and 39, respectively);

D) huAb13v4 (VH amino acid sequence set forth in SEQ ID NO: 146 and VHCDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 33,34, and 35, respectively; and VL amino acid sequence set forth in SEQ IDNO: 145 and VL CDR1, CDR2, and CDR3 amino acid sequences set forth inSEQ ID NOs: 37, 38, and 39, respectively);

E) huAb13v5 (VH amino acid sequence set forth in SEQ ID NO: 147 and VHCDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 33,34, and 35, respectively; and VL amino acid sequence set forth in SEQ IDNO: 143 and VL CDR1, CDR2, and CDR3 amino acid sequences set forth inSEQ ID NOs: 37, 38, and 39, respectively);

F) huAb13 v6 (VH amino acid sequence set forth in SEQ ID NO: 147 and VHCDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 33,34, and 35, respectively; and VL amino acid sequence set forth in SEQ IDNO: 145 and VL CDR1, CDR2, and CDR3 amino acid sequences set forth inSEQ ID NOs: 37, 38, and 39, respectively);

G) huAb13v7 (VH amino acid sequence set forth in SEQ ID NO: 148 and VHCDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 33,34, and 35, respectively; and VL amino acid sequence set forth in SEQ IDNO: 143 and VL CDR1, CDR2, and CDR3 amino acid sequences set forth inSEQ ID NOs: 37, 38, and 39, respectively);

H) huAb13v8 (VH amino acid sequence set forth in SEQ ID NO: 148 and VHCDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ NOs: 33, 34,and 35, respectively; and VL amino acid sequence set forth in SEQ ID NO:144 and VL CDR1, CDR2, and CDR3 amino Acid sequences set forth in SEQ IDNOs: 37, 38, and 39, respectively);

I) huAb13v9 (VH amino acid sequence set forth in SEQ ID NO: 148 and VHCDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 33,34, and 35, respectively; and VL amino acid sequence set forth in SEQ IDNO: 145 and VL CDR1, CDR2, and CDR3 amino acid sequences set forth inSEQ ID NOs: 37, 38, and 39, respectively).

Thus, in one aspect the present invention provides antibodies comprisingvariable and/or CDR sequences from a humanized antibody derived fromchAb13. In one embodiment, the invention features anti-B7-H3 antibodieswhich are derived from chAb13 have improved characteristics, e.g.,improved binding affinity to isolated B7-H3 protein and improved bindingto B7-H3 expressing cells, as described in the Examples below.Collectively these novel antibodies are referred to herein as “Ab13variant antibodies.” Generally, the Ab13 variant antibodies retain thesame epitope specificity as Ab13. In various embodiments, anti-B7-H3antibodies, or antigen binding fragments thereof, of the invention arecapable of modulating a biological function of B7-H3.

In one aspect, the present invention provides a humanized antibody, orantigen binding portion thereof, having a heavy chain variable regionincluding an amino acid sequence set forth in SEQ ID NOs: 146, 147, or148; and/or a light chain variable region including an amino acidsequence set forth in SEQ ID NOs: 143, 144, or 145.

In another aspect, the present invention is directed to an anti-B7-H3antibody, or antigen binding portion thereof, of the invention comprisesa heavy chain variable region comprising a CDR1 domain comprising anamino acid sequence as set forth in SEQ ID NO: 33; a CDR2 domaincomprising an amino acid sequence as set forth in SEQ ID NO: 34; and aCDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO:35; and a light chain variable region comprising a CDR1 domaincomprising an amino acid sequence as set forth in SEQ ID NO: 37; a CDR2domain comprising an amino acid sequence as set forth in SEQ ID NO: 38;and a CDR3 domain comprising an amino acid sequence as set forth in SEQID NO: 39.

In one aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 147, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 144. In one embodiment, the inventionprovides an anti-B7H3 antibody comprising the CDR sequences set forth inthe variable regions of huAb13v1 (SEQ ID NOs. 144 and 147).

In one aspect, the present invention is directed to an anti-B7-H3antibody, or antigen binding portion thereof, having a heavy chaincomprising the amino acid sequence of SEQ ID NO: 168 and a light chaincomprising the amino acid sequence of SEQ ID NO: 169.

In one aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 146, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 143.

In one aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 146, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 144.

In one aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 146, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 145.

In one aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 147, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 143.

In one aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 147, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 145.

In one aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 148, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 143.

In one aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 148, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 144.

In one aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 148, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 145.

Humanized Anti-B7-H3 Antibodies Derived from chAb18

The ten different humanized antibodies created based on chAb18 includethe following:

A) huAb18v1 (VH amino acid sequence set forth in SEQ ID NO: 116 and VHCDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 25,26, and 27, respectively; and VL amino acid sequence set forth in SEQ IDNO: 120 and VL CDR1, CDR2, and CDR3 amino acid sequences set forth inSEQ ID NOs: 29, 30, and 31, respectively);

B) huAb18v2 (VH amino acid sequence set forth in SEQ ID NO: 118 and VHCDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 25,119, and 27, respectively; and VL amino acid sequence set forth in SEQID NO: 120 and VL CDR1, CDR2, and CDR3 amino acid sequences set forth inSEQ ID NOs: 29, 30, and 31, respectively);

C) huAb18v3 (VH amino acid sequence set forth in SEQ ID NO: 117 and VHCDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 25,26, and 27, respectively; and VL amino acid sequence set forth in SEQ IDNO: 121 and VL CDR1, CDR2, and CDR3 amino acid sequences set forth inSEQ ID NOs: 29, 30, and 31, respectively);

D) huAb18v4 (VH amino acid sequence set forth in SEQ ID NO: 118 and VHCDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 25,119, and 27, respectively; and VL amino acid sequence set forth in SEQID NO: 121 and VL CDR1, CDR2, and CDR3 amino acid sequences set forth inSEQ ID NOs: 29, 30, and 31, respectively);

E) huAb18v5 (VH amino acid sequence set forth in SEQ ID NO: 116 and VHCDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 25,26, and 27, respectively; and VL amino acid sequence set forth in SEQ IDNO: 123 and VL CDR1, CDR2, and CDR3 amino acid sequences set forth inSEQ ID NOs: 29, 30, and 31, respectively);

F) huAb18v6 (VH amino acid sequence set forth in SEQ ID NO: 118 and VHCDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ NOs: 25, 119,and 27, respectively; and VL amino acid sequence set forth in SEQ ID NO:123 and VL CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ IDNOs: 29, 30, and 31, respectively);

G) huAb18v7 (VH amino acid sequence set forth in SEQ ID NO: 118 and VHCDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 25,119, and 27, respectively; and VL amino acid sequence set forth in SEQID NO: 124 and VL CDR1, CDR2, and CDR3 amino acid sequences set forth inSEQ ID NOs: 29, 30, and 31, respectively);

H) huAb18v8 (VH amino acid sequence set forth in SEQ ID NO: 117 and VHCDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 25,26, and 27, respectively; and VL amino acid sequence set forth in SEQ IDNO: 122 and VL CDR1, CDR2, and CDR3 amino acid sequences set forth inSEQ ID NOs: 29, 30, and 31, respectively);

I) huAb18v9 (VH amino acid sequence set forth in SEQ ID NO: 117 and VHCDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 25,26, and 27, respectively; and VL amino acid sequence set forth in SEQ IDNO: 124 and VL CDR1, CDR2, and CDR3 amino acid sequences set forth inSEQ ID NOs: 29, 30, and 31, respectively); and

J) huAb18v10 (VH amino acid sequence set forth in SEQ ID NO: 118 and VHCDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 25,119, and 27, respectively; and VL amino acid sequence set forth in SEQID NO: 122 and VL CDR1, CDR2, and CDR3 amino acid sequences set forth inSEQ ID NOs: 29, 30, and 31, respectively).

Thus, in one aspect the present invention provides antibodies comprisingvariable and/or CDR sequences from a humanized antibody derived fromchAb18. In one embodiment, the invention features anti-B7-H3 antibodieswhich are derived from Ab18 have improved characteristics, e.g.,improved binding affinity to isolated B7-H3 protein and improved bindingto B7-H3 expressing cells, as described in the Examples below.Collectively these novel antibodies are referred to herein as “Ab18variant antibodies.” Generally, the Ab18 variant antibodies retain thesame epitope specificity as Ab18. In various embodiments, anti-B7-H3antibodies, or antigen binding fragments thereof, of the invention arecapable of modulating a biological function of B7-H3.

In one aspect, the present invention provides a humanized antibody, orantigen binding portion thereof, having a heavy chain variable regionincluding an amino acid sequence set forth in SEQ ID NOs: 116, 117, or118; and/or a light chain variable region including an amino acidsequence set forth in SEQ ID NOs: 120, 121, 122, 123 or 124.

In another aspect, the present invention is directed to an anti-B7-H3antibody, or antigen binding portion thereof, of the invention comprisesa heavy chain variable region comprising a CDR1 domain comprising anamino acid sequence as set forth in SEQ ID NO: 25; a CDR2 domaincomprising an amino acid sequence as set forth in SEQ ID NO: 26 or 119;and a CDR3 domain comprising an amino acid sequence as set forth in SEQID NO: 27; and a light chain variable region comprising a CDR1 domaincomprising an amino acid sequence as set forth in SEQ ID NO: 29; a CDR2domain comprising an amino acid sequence as set forth in SEQ ID NO: 30;and a CDR3 domain comprising an amino acid sequence as set forth in SEQID NO: 31.

In one aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 116, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 120.

In another aspect, the present invention is directed to a humanizedanti-B7-H3 antibody, or antigen-binding portion thereof, having a heavychain variable domain region including (a) a CDR1 having an amino acidsequence as set forth in SEQ ID NO: 25; (b) a CDR2 having an amino acidsequence as set forth in SEQ ID NO: 26; and (c) a CDR3 having an aminoacid sequence as set forth in SEQ ID NO: 27; and a light chain variableregion including (a) a CDR1 having an amino acid sequence as set forthin SEQ ID NO: 29; (b) a CDR2 having an amino acid sequence as set forthin SEQ ID NO: 30; and (c) a CDR3 having an amino acid sequence as setforth in SEQ ID NO: 31.

In one aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 118, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 120.

In another aspect, the present invention is directed to a humanizedanti-B7-H3 antibody, or antigen-binding portion thereof, having a heavychain variable domain region including (a) a CDR1 having an amino acidsequence as set forth in SEQ ID NO: 25; (b) a CDR2 having an amino acidsequence as set forth in SEQ ID NO: 119; and (c) a CDR3 having an aminoacid sequence as set forth in SEQ ID NO: 27; and a light chain variableregion including (a) a CDR1 having an amino acid sequence as set forthin SEQ ID NO: 29; (b) a CDR2 having an amino acid sequence as set forthin SEQ ID NO: 30; and (c) a CDR3 having an amino acid sequence as setforth in SEQ ID NO: 31.

In one aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 117, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 121.

In one aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 118, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 121.

In one aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 116, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 123.

In one aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 118, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 123.

In one aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 118, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 124.

In one aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 117, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 122.

In one aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 117, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 124.

In one aspect, the present invention is directed to an anti-B7-H3antibody, or antigen-binding portion thereof, having a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNO: 118, and a light chain variable region including an amino acidsequence set forth in SEQ ID NO: 122.

In one aspect, the present invention provides a humanized antibody, orantigen binding portion thereof having a heavy chain variable regionincluding an amino acid sequence set forth in SEQ ID NOs: 116, 117, 118,146, 147, 148, 125, 126, 127, 131, 139, or 141; and/or a light chainvariable region including an amino acid sequence set forth in SEQ IDNOs: 120, 121, 122, 123, 124, 143, 144, 145, 128, 129, 130, 133, 135, or137.

In another aspect, the present invention is directed to an anti-B7-H3antibody, or antigen binding portion thereof, of the invention comprisesa heavy chain variable region comprising a CDR1 domain comprising anamino acid sequence as set forth in SEQ ID NO: 10, 25, or 33; a CDR2domain comprising an amino acid sequence as set forth in SEQ ID NO: 11,26, 34, 119, 132, 140, or 142; and a CDR3 domain comprising an aminoacid sequence as set forth in SEQ ID NO: 12, 27, or 35; and a lightchain variable region comprising a CDR1 domain comprising an amino acidsequence as set forth in SEQ ID NO: 14, 29, 37, 134, 136, or 138; a CDR2domain comprising an amino acid sequence as set forth in SEQ ID NO: 7,30, or 38; and a CDR3 domain comprising an amino acid sequence as setforth in SEQ ID NO: 15, 31 or 39.

In another aspect, the invention provides an anti-B7-H3 antibody, orantigen binding fragment thereof, that specifically competes with ananti-B7-H3 antibody, or fragment thereof, as described herein, whereinsaid competition can be detected in a competitive binding assay usingsaid antibody, the human B7-H3 polypeptide, and the anti-B7-H3 antibodyor fragment thereof.

In particular embodiments, the competing antibody, or antigen bindingportion thereof, is an antibody, or antigen binding portion thereof,that competes with huAb3v2.5, huAb3v2.6, or huAb13 v1.

In one embodiment, the anti-B7-H3 antibodies, or antigen bindingportions thereof, of the invention bind to the extracellular domain ofhuman B7-H3 (SEQ ID NO: 152) with a dissociation constant (K_(D)) ofabout 1×10⁻⁶ M or less, as determined by surface plasmon resonance.Alternatively, the antibodies, or antigen binding portions thereof, bindto human B7-H3 with a K_(D) of between about 1×10⁻⁶M and about 1×10⁻¹¹M, as determined by surface plasmon resonance. In a further alternative,antibodies, or antigen binding portions thereof, bind to human B7-H3with a K_(D) of between about 1×10⁻⁶ M and about 1×10⁻⁷ M, as determinedby surface plasmon resonance. Alternatively, antibodies, or antigenbinding portions thereof, of the invention binds to human B7-H3 with aK_(D) of between about 1×10⁻⁶ M and about 5×10⁻¹¹ M, about 1×10⁻⁶ M andabout 5×10⁻¹⁰ M; a K_(D) of between about 1×10⁻⁶ M and about 1×10⁻⁹ M; aK_(D) of between about 1×10⁻⁶ M and about 5×10⁻⁹M; a K_(D) of betweenabout 1×10⁻⁶ M and about 1×10⁻⁸M; a K_(D) of between about 1×10⁻⁶ M andabout 5×10⁻⁸M; a K_(D) of between about 1×10⁻⁷ M and about 3.4×10⁻¹¹M; aK_(D) of between about 5.9×10⁻⁷ M; and about 2.2×10⁻⁷ M, as determinedby surface plasmon resonance.

In one embodiment, the antibodies, or antigen binding portions thereofof the invention bind to human B7-H3 (SEQ ID NO: 149) with a K_(D) ofabout 1×10⁻⁶M or less, as determined by surface plasmon resonance.Alternatively, the antibodies, or antigen binding portions thereof, ofthe invention bind to human B7-H3 (SEQ ID NO: 149) with a K_(D) ofbetween about 8.2×10⁻⁹ M and about 6.3×10⁻¹⁰M; a K_(D) of between about8.2×10⁻⁹M and about 2.0×10⁻⁹M; a K_(D) of between about 2.3×10⁻⁹M andabout 1.5×10⁻¹⁰ M, as determined by surface plasmon resonance.

The foregoing establish a novel family of B7-H3 binding proteins,isolated in accordance with this invention, and including antigenbinding polypeptides that comprise the CDR sequences listed in theSequence Table provided herein.

Anti-B7-H3 antibodies provided herein may comprise a heavy chainvariable region comprising CDR1, CDR2 and CDR3 sequences and a lightchain variable region comprising CDR1, CDR2 and CDR3 sequences, whereinone or more of these CDR sequences comprise specified amino acidsequences based on the antibodies described herein (e.g., huAb13v1 orhuAb3v2.5), or conservative modifications thereof and wherein theantibodies retain the desired functional properties of theanti-B7-H3antibodies described herein. Accordingly, the anti-B7-H3antibody, or antigen binding portion thereof, may comprise a heavy chainvariable region comprising CDR1, CDR2, and CDR3 sequences and a lightchain variable region comprising CDR1, CDR2, and CDR3 sequences,wherein: (a) the heavy chain variable region CDR3 sequence comprises SEQID NO: 12 or 35, and conservative modifications thereof, e.g., 1, 2, 3,4, 5, 1-2, 1-3, 1-4 or 1-5 conservative amino acid substitutions; (b)the light chain variable region CDR3 sequence comprises SEQ ID NO: 15 or39, and conservative modifications thereof, e.g., 1, 2, 3, 4, 5, 1-2,1-3, 1-4 or 1-5 conservative amino acid substitutions; (c) the antibodyspecifically binds to B7-H3, and (d) the antibody exhibits 1, 2, 3, 4,5, 6, or all of the following functional properties described herein,e.g., binding to soluble human B7-H3. In a one embodiment, the heavychain variable region CDR2 sequence comprises SEQ ID NO: 140 or 34, andconservative modifications thereof, e.g., 1, 2, 3, 4, 5, 1-2, 1-3, 1-4or 1-5 conservative amino acid substitutions; and the light chainvariable region CDR2 sequence comprises SEQ ID NO: 7 or 38, andconservative modifications thereof, e.g., 1, 2, 3, 4, 5, 1-2, 1-3, 1-4or 1-5 conservative amino acid substitutions. In another preferredembodiment, the heavy chain variable region CDR1 sequence comprises SEQID NO: 10 or 33, and conservative modifications thereof, e.g., 1, 2, 3,4, 5, 1-2, 1-3, 1-4 or 1-5 conservative amino acid substitutions; andthe light chain variable region CDR1 sequence comprises SEQ ID NO: 136,138, or 37, and conservative modifications thereof, e.g., 1, 2, 3, 4, 5,1-2, 1-3, 1-4 or 1-5 conservative amino acid substitutions.

Conservative amino acid substitutions may also be made in portions ofthe antibodies other than, or in addition to, the CDRs. For example,conservative amino acid modifications may be made in a framework regionor in the Fe region. A variable region or a heavy or light chain maycomprise 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or1-50 conservative amino acid substitutions relative to the anti-B7-H3antibody sequences provided herein. In certain embodiments, theanti-97-H3antibody comprises a combination of conservative andnon-conservative amino acid modification.

To generate and to select CDRs having preferred B7-H3 binding and/orneutralizing activity with respect to hB7-H3, standard methods known inthe art for generating antibodies, or antigen binding portions thereof,and assessing the B7-H3 binding and/or neutralizing characteristics ofthose antibodies, or antigen binding portions thereof, may be used,including but not limited to those specifically described herein.

In certain embodiments, the antibody comprises a heavy chain constantregion, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgDconstant region. In certain embodiments, the anti-B7-113 antibody, orantigen binding portion thereof, comprises a heavy chain immunoglobulinconstant domain selected from the group consisting of a human IgGconstant domain, a human IgM constant domain, a human IgE constantdomain, and a human IgA constant domain. In further embodiments, theantibody, or antigen binding portion thereof, has an IgG1 heavy chainconstant region, an IgG2 heavy chain constant region, an IgG3 constantregion, or an IgG4 heavy chain constant region. Preferably, the heavychain constant region is an IgG1 heavy chain constant region or an IgG4heavy chain constant region. Furthermore, the antibody can comprise alight chain constant region, either a kappa light chain constant regionor a lambda light chain constant region. Preferably, the antibodycomprises a kappa light chain constant region. Alternatively, theantibody portion can be, for example, a Fab fragment or a single chainFv fragment.

In certain embodiments, the anti-B7-H3 antibody binding portion is aFab, a Fab′, a F(ab′)2, Fv, a disulfide linked Fv, an scFv, a singledomain antibody, or a diabody.

In certain embodiments, the anti-B7-H3 antibody, or antigen bindingportion thereof, is a multispecific antibody, e.g. a bispecificantibody.

Replacements of amino acid residues in the Fc portion to alter antibodyeffector function have been described (Winter, et al. U.S. Pat. Nos.5,648,260 and 5,624,821, incorporated by reference herein). The Fcportion of an antibody mediates several important effector functionse.g. cytokine induction, ADCC, phagocytosis, complement dependentcytotoxicity (CDC) and half-life/clearance rate of antibody andantigen-antibody complexes. In some cases these effector functions aredesirable for therapeutic antibody but in other cases might beunnecessary or even deleterious, depending on the therapeuticobjectives. Certain human IgG isotypes, particularly IgG1 and IgG3,mediate ADCC and CDC via binding to FcγRs and complement C1q,respectively. Neonatal Fe receptors (FcRn) are the critical componentsdetermining the circulating half-life of antibodies. In still anotherembodiment at least one amino acid residue is replaced in the constantregion of the antibody, for example the Fc region of the antibody, suchthat effector functions of the antibody are altered. One embodiment ofthe invention includes a recombinant chimeric antigen receptor (CAR)comprising the binding regions of the antibodies described herein, e.g.,the heavy and/or light chain CDRs of huAb13v1. A recombinant CAR, asdescribed herein, may be used to redirect T cell specificity to anantigen in a human leukocyte antigen (HLA)-independent fashion, Thus,CARS of the invention may be used in immunotherapy to help engineer ahuman subject's own immune cells to recognize and attack the subject'stumor (see, e.g., U.S. Pat. Nos. 6,410,319; 8,389,282; 8,822,647;8,906,682; 8,911,993; 8,916,381; 8,975,071; and U.S. Patent Appln. Publ.No. US20140322275, each of which is incorporated by reference hereinwith respect to CAR technology). This type of immunotherapy is calledadoptive cell transfer (ACT), and may be used to treat cancer in asubject in need thereof.

An anti-B7-H3 CAR of the invention preferably contains a extracellularantigen-binding domain specific for B7-H3, a transmembrane domain whichis used to anchor the CAR into a T cell, and one or more intracellularsignaling domains. In one embodiment of the invention, the CAR includesa transmembrane domain that comprises a transmembrane domain of aprotein selected from the group consisting of the alpha, beta or zetachain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8,CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.In one embodiment of the invention, the CAR comprises a costimulatorydomain, e.g., a costimulatory domain comprising a functional signalingdomain of a protein selected from, the group consisting of OX40, CD2,CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), and 4-1BB(CD13). In certain embodiments of the invention, the CAR comprises anscFv comprising the CDR or variable regions described herein e.g., CDRsor variable regions from the huAb13v1 antibody, a transmembrane domain,a co-stimulatory domain (e.g., a functional signaling domain from CD28or 4-1BB), and a signaling domain comprising a functional signalingdomain from CD3 (e.g., CD3-zeta).

In certain embodiments, the invention includes a T cell comprising a CAR(also referred to as a CART cell) comprising antigen binding regions,e.g. CDRs, of the antibodies described herein or an scFv describedherein.

In certain embodiments of the invention, the CAR comprises a heavy chainvariable region comprising a CDR1 domain comprising an amino acidsequence as set forth in SEQ ID NO: 10, 25, or 33; a CDR2 domaincomprising an amino acid sequence as set forth in SEQ ID NO: 11, 26, 34,119, 132, 140, or 142; and a CDR3 domain comprising an amino acidsequence as set forth in SEQ ID NO: 12, 27, or 35; and a light chainvariable region comprising a CDR1 domain comprising an amino acidsequence as set forth in SEQ ID NO: 14, 29, 37, 134, 136, or 138; a CDR2domain comprising an amino acid sequence as set forth in SEQ ID NO: 7,30, or 38; and a CDR3 domain comprising an amino acid sequence as setforth in SEQ ID NO: 15, 31 or 39.

In certain embodiments of the invention, the CAR comprises a heavy chainvariable domain region including (a) a CDR1 having an amino acidsequence as set forth in SEQ ID NO: 10; (b) a CDR2 having an amino acidsequence as set forth in SEQ ID NO: 11; and (c) a CDR3 having an aminoacid sequence as set forth in SEQ ID NO: 12; and a light chain variableregion including (a) a CDR1 having an amino acid sequence as set forthin SEQ ID NO: 14; (b) a CDR2 having an amino acid sequence as set forthin SEQ ID NO: 7; and (c) a CDR3 having an amino acid sequence as setforth in SEQ ID NO: 15.

In certain embodiments of the invention, the CAR comprises a heavy chainvariable domain region including (a) a CDR1 having an amino acidsequence as set forth in SEQ ID NO: 10; (b) a CDR2 having an amino acidsequence as set forth in SEQ ID NO: 132; and (c) a CDR3 having an aminoacid sequence as set forth in SEQ ID NO: 12; and a light chain variableregion including (a) a CDR1 having an amino acid sequence as set forthin SEQ ID NO: 134; (b) a CDR2 having an amino acid sequence as set forthin SEQ ID NO: 7; and (c) a CDR3 having an amino acid sequence as setforth in SEQ ID NO: 15.

In certain embodiments of the invention, the CAR comprises a heavy chainvariable domain region including (a) a CDR1 having an amino acidsequence as set forth in SEQ ID NO: 10; (b) a CDR2 having an amino acidsequence as set forth in SEQ ID NO: 132; and (c) a CDR3 having an aminoacid sequence as set forth in SEQ ID NO: 12; and a light chain variableregion including (a) a CDR1 having an amino acid sequence as set forthin SEQ ID NO: 136; (b) a CDR2 having an amino acid sequence as set forthin SEQ ID NO: 7; and (c) a CDR3 having an amino acid sequence as setforth in SEQ ID NO: 15.

In certain embodiments of the invention, the CAR comprises a heavy chainvariable domain region including (a) a CDR1 having an amino acidsequence as set forth in SEQ ID NO: 10; (h) a CDR2 having an amino acidsequence as set forth in SEQ ID NO: 132; and (c) a CDR3 having an aminoacid sequence as set forth in SEQ ID NO: 12; and a light chain variableregion including (a) a CDR1 having an amino acid sequence as set forthin SEQ ID NO: 138; (b) a CDR2 having an amino acid sequence as set forthin SEQ ID NO: 7; and (c) a CDR3 having an amino acid sequence as setforth in SEQ ID NO: 15.

In certain embodiments of the invention, the CAR comprises a heavy chainvariable domain region including (a) a CDR1 having an amino acidsequence as set forth in SEQ ID NO: 10; (h) a CDR2 having an amino acidsequence as set forth in SEQ ID NO: 140; and (c) a CDR3 having an aminoacid sequence as set forth in SEQ ID NO: 12; and a light chain variableregion including (a) a CDR1 having an amino acid sequence as set forthin SEQ ID NO: 134; (b) a CDR2 having an amino acid sequence as set forthin SEQ ID NO: 7; and (c) a CDR3 having an amino acid sequence as setforth in SEQ ID NO: 15.

In certain embodiments of the invention, the CAR comprises a heavy chainvariable domain region including (a) a CDR1 having an amino acidsequence as set forth in SEQ ID NO: 10; (h) a CDR2 having an amino acidsequence as set forth in SEQ ID NO: 140; and (c) a CDR3 having an aminoacid sequence as set forth in SEQ ID NO: 12; and a light chain variableregion including (a) a CDR1 having an amino acid sequence as set forthin SEQ ID NO: 136; (b) a CDR2, having an amino acid sequence as setforth in SEQ ID NO: 7; and (c) a CDR3 having an amino acid sequence asset forth in SEQ ID NO: 15.

In certain embodiments of the invention, the CAR comprises a heavy chainvariable domain region including (a) a CDR1 having an amino acidsequence as set forth in SEQ ID NO: 10; (b) a CDR2 having an amino acidsequence as set forth in SEQ ID NO: 140; and (c) a CDR3 having an aminoacid sequence as set forth in SEQ ID NO: 12; and a light chain variableregion including (a) a CDR1 having an amino acid sequence as set forthin SEQ ID NO: 138; (b) a CDR2 having an amino acid sequence as set forthin SEQ ID NO: 7; and (c) a CDR3 having an amino acid sequence as setforth in SEQ ID NO: 15.

In certain embodiments of the invention, the CAR comprises a heavy chainvariable domain region including (a) a CDR1 having an amino acidsequence as set forth in SEQ ID NO: 10; (b) a CDR2 having an amino acidsequence as set forth in SEQ ID NO: 142; and (c) a CDR3 having an aminoacid sequence as set forth in SEQ ID NO: 12; and a light chain variableregion including (a) a CDR1 having an amino acid sequence as set forthin SEQ ID NO: 134; (h) a CDR2 having an amino acid sequence as set forthin SEQ ID NO: 7; and (c) a CDR3 having an amino acid sequence as setforth in SEQ ID NO: 15.

In certain embodiments of the invention, the CAR comprises a heavy chainvariable domain region including (a) a CDR1 having an amino acidsequence as set forth in SEQ ID NO: 10; (b) a CDR2 having an amino acidsequence as set forth in SEQ ID NO: 142; and (c) a CDR3 having an aminoacid sequence as set forth in SEQ ID NO: 12; and a light chain variableregion including (a) a CDR1 having an amino acid sequence as set forthin SEQ ID NO: 136; (b) a CDR2 having an amino acid sequence as set forthin SEQ ID NO: 7; and (c) a CDR3 having an amino acid sequence as setforth in SEQ ID NO: 15.

In certain embodiments of the invention, the CAR comprises a heavy chainvariable domain region including (a) a CDR1 having an amino acidsequence as set forth in SEQ ID NO: 10; (b) a CDR2 having an amino acidsequence as set forth in SEQ ID NO: 142; and (c) a CDR3 having an aminoacid sequence as set forth in SEQ ID NO: 12; and a light chain variableregion including (a) a CDR1 having an amino acid sequence as set forthin SEQ ID NO: 138; (b) a CDR2 having an amino acid sequence as set forthin SEQ ID NO: 7; and (c) a CDR3 having an amino acid sequence as setforth in SEQ. ID NO: 15.

In certain embodiments of the invention, the CAR comprises a heavy chainvariable region comprising a CDR1 domain comprising an amino acidsequence as set forth in SEQ ID NO: 33; a CDR2 domain comprising anamino acid sequence as set forth in SEQ ID NO: 34; and a CDR3 domaincomprising an amino acid sequence as set forth in SEQ ID NO: 35; and alight chain variable region comprising a CDR1 domain comprising an aminoacid sequence as set forth in SEQ ID NO: 37; a CDR2 domain comprising anamino acid sequence as set forth in SEQ ID NO: 38; and a CDR3 domaincomprising an amino acid sequence as set forth in SEQ ID NO: 39.

In certain embodiments of the invention, the CAR comprises a heavy chainvariable region comprising a CDR1 domain comprising an amino acidsequence as set forth in SEQ ID NO: 25; a CDR2 domain comprising anamino acid sequence as set forth in SEQ ID NO: 26 or 119; and a CDR3domain comprising an amino acid sequence as set forth in SEQ ID NO: 27;and a light chain variable region comprising a CDR1 domain comprising anamino acid sequence as set forth in SEQ ID NO: 29; a CDR2 domaincomprising an amino acid sequence as set forth in SEQ ID NO: 30; and aCDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO:31.

In certain embodiments of the invention, the CAR comprises a heavy chainvariable domain region including (a) a CDR1 having an amino acidsequence as set forth in SEQ ID NO: 25; (b) a CDR2 having an amino acidsequence as set forth in SEQ ID NO: 26; and (c) a CDR3 having an aminoacid sequence as set forth in SEQ ID NO: 27; and a light chain variableregion including (a) a CDR1 having an amino acid sequence as set forthin SEQ ID NO: 29; (b) a CDR2 having an amino acid sequence as set forthin SEQ ID NO: 30; and (c) a CDR3 having an amino acid sequence as setforth in SEQ ID NO: 31.

In certain embodiments of the invention, the CAR comprises a heavy chainvariable domain region including (a) a CDR1 having an amino acidsequence as set forth in SEQ ID NO: 25; (b) a CDR2 having an amino acidsequence as set forth in SEQ ID NO: 19; and (c) a CDR3 having an aminoacid sequence as set forth in SEQ ID NO: 27; and a light chain variableregion including (a) a CDR1 having an amino acid sequence as set forthin SEQ ID NO: 29; (b) a CDR2 having an amino acid sequence as set forthin SEQ ID NO: 30; and (c) a CDR3 having an amino acid sequence as setforth in SEQ ID NO: 31.

One embodiment of the invention includes a labeled anti-B7-H3 antibody,or antibody portion thereof, where the antibody is derivatized or linkedto one or more functional molecule(s) (e.g another peptide or protein).For example, a labeled antibody can be derived by functionally linkingan antibody or antibody portion of the invention (by chemical coupling,genetic fusion, noncovalent association or otherwise) to one or moreother molecular entities, such as another antibody (e.g., a bispecificantibody or a diabody), a detectable agent, a pharmaceutical agent, aprotein or peptide that can mediate the association of the antibody orantibody portion with another molecule (such as a streptavidin coreregion or a polyhistidine tag), and/or a cytotoxic or therapeutic agentselected from the group consisting of a mitotic inhibitor, an antitumorantibiotic, an immunomodulating agent, a vector for gene therapy, analkylating agent, an antiangiogenic agent, an antimetabolite, aboron-containing agent, a chemoprotective agent, a hormone, anantihormone agent, a corticosteroid, a photoactive therapeutic agent, anoligonucleotide, a radionuclide agent, a topoisomerase inhibitor, akinase inhibitor, a radiosensitizer, and a combination thereof.

Useful detectable agents with which an antibody or antibody portionthereof, may be derivatized include fluorescent compounds. Exemplaryfluorescent detectable agents include fluorescein, fluoresceinisothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonylchloride, phycoerythrin and the like. An antibody may also bederivatized with detectable enzymes, such as alkaline phosphatase,horseradish peroxidase, glucose oxidase and the like. When an antibodyis derivatized with a detectable enzyme, it is detected by addingadditional reagents that the enzyme uses to produce a detectablereaction product. For example, when the detectable agent horseradishperoxidase is present the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which isdetectable. An antibody may also be derivatized with biotin, anddetected through indirect measurement of avidin or streptavidin binding.

In one embodiment, the antibody of the invention is conjugated to animaging agent, Examples of imaging agents that may be used in thecompositions and methods described herein include, but are not limitedto, a radiolabel (e.g., indium), an enzyme, a fluorescent label, aluminescent label, a bioluminescent label, a magnetic label, and biotin.

In one embodiment, the antibodies or ADCs are linked to a radiolabel,such as, but not limited to, indium (¹¹¹In). ¹¹¹Indium may be used tolabel the antibodies and ADCs described herein for use in identifyingB7-H3 positive tumors. In a certain embodiment, anti-B7-H3 antibodies(or ADCs) described herein are labeled with via a bifunctional chelatorwhich is a bifunctional cyclohexyl diethylenetriaminepentaacetic acid(DTPA) chelate (see U.S. Pat. Nos. 5,124,471; 5,434,287; and 5,286,850,each of which is incorporated herein by reference).

Another embodiment of the invention provides a glycosylated bindingprotein wherein the anti-B7-H3 antibody or antigen binding portionthereof comprises one or more carbohydrate residues. Nascent in vivoprotein production may undergo further processing, known aspost-translational modification. In particular, sugar (glycosyl)residues may be added enzymatically, a process known as glycosylation.The resulting proteins bearing covalently linked oligosaccharide sidechains are known as glycosylated proteins or glycoproteins. Antibodiesare glycoproteins with one or more carbohydrate residues in the Fedomain, as well as the variable domain. Carbohydrate residues in the Fedomain have important effect on the effector function of the Fc domain,with minimal effect on antigen binding or half-life of the antibody (R.Jefferis, Biotechnol. Prog. 21 (2005), pp. 11-16), In contrast,glycosylation of the variable domain may have an effect on the antigenbinding activity of the antibody. Glycosylation in the variable domainmay have a negative effect on antibody binding affinity, likely due tosteric hindrance (Co, M. S., et al., Mol. Immunol. (1993) 30:1361-1367),or result in increased affinity for the antigen (Wallick, S. C., et al.,Exp. Med (1988) 168:1099-1109; Wright, A., et al., EMBO J. (1991)10:2717-2723).

One aspect of the invention is directed to generating glycosylation sitemutants in which the O- or N-linked glycosylation site of the bindingprotein has been mutated. One skilled in the art can generate suchmutants using standard well-known technologies. Glycosylation sitemutants that retain the biological activity, but have increased ordecreased binding activity, are another object of the invention.

In still another embodiment, the glycosylation of the anti-B7-H3antibody or antigen binding portion of the invention is modified. Forexample, an aglycoslated antibody can be made (i.e., the antibody lacksglycosylation). Glycosylation can be altered to, for example, increasethe affinity of the antibody for antigen. Such carbohydratemodifications can be accomplished by, for example, altering one or moresites of glycosylation within the antibody sequence. For example, one ormore amino acid substitutions can be made that result in elimination ofone or more variable region glycosylation sites to thereby eliminateglycosylation at that site. Such aglycosylation may increase theaffinity of the antibody for antigen. Such an approach is described infurther detail in PCT Publication WO2003016466A2, and U.S. Pat. Nos.5,714,350 and 6,350,861, each of which is incorporated herein byreference in its entirety.

Additionally or alternatively, a modified anti-B7-H3 antibody of theinvention can be made that has an altered type of glycosylation, such asa hypofucosylated antibody having reduced amounts of fucosyl residues oran antibody having increased bisecting GlcNAc structures. Such alteredglycosylation patterns have been demonstrated to increase the ADCCability of antibodies. Such carbohydrate modifications can beaccomplished by, for example, expressing the antibody in a host cellwith altered glycosylation machinery. Cells with altered glycosylationmachinery have been described in the art and can be used as host cellsin which to express recombinant antibodies of the invention to therebyproduce an antibody with altered glycosylation. See, for example,Shields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740; Umana etal. (1999) Nat. Biotech. 17:176-1, as well as, European Patent No: EP1,176,195; PCT Publications WO 03/035835; WO 99/54342 80, each of whichis incorporated herein by reference in its entirety.

Protein glycosylation depends on the amino acid sequence of the proteinof interest, as well as the host cell in which the protein is expressed.Different organisms may produce different glycosylation enzymes (e.g.,glycosyltransferases and glycosidases), and have different substrates(nucleotide sugars) available. Due to such factors, proteinglycosylation pattern, and composition of glycosyl residues, may differdepending on the host system in which the particular protein isexpressed. Glycosyl residues useful in the invention may include, butare not limited to, glucose, galactose, mannose, fucose,n-acetylglucosamine and sialic acid. Preferably the glycosylated bindingprotein comprises glycosyl residues such that the glycosylation patternis human.

Differing protein glycosylation may result in differing proteincharacteristics. For instance, the efficacy of a therapeutic proteinproduced in a microorganism host, such as yeast, and glycosylatedutilizing the yeast endogenous pathway may be reduced compared to thatof the same protein expressed in a mammalian cell, such as a CHO cellline. Such glycoproteins may also be immunogenic in humans and showreduced half-life in vivo after administration. Specific receptors inhumans and other animals may recognize specific glycosyl residues andpromote the rapid clearance of the protein from the bloodstream. Otheradverse effects may include changes in protein folding, solubility,susceptibility to proteases, trafficking, transport,compartmentalization, secretion, recognition by other proteins orfactors, antigenicity, or allergenicity. Accordingly, a practitioner mayprefer a therapeutic protein with a specific composition and pattern ofglycosylation, for example glycosylation composition and patternidentical, or at least similar, to that produced in human cells or inthe species-specific cells of the intended subject animal.

Expressing glycosylated proteins different from that of a host cell maybe achieved by genetically modifying the host cell to expressheterologous glycosylation enzymes. Using recombinant techniques, apractitioner may generate antibodies or antigen binding portions thereofexhibiting human protein glycosylation. For example, yeast strains havebeen genetically modified to express non-naturally occurringglycosylation enzymes such that glycosylated proteins (glycoproteins)produced in these yeast strains exhibit protein glycosylation identicalto that of animal cells, especially human cells (U.S. patent PublicationNos. 20040018590 and 20020137134 and PCT publication WO2005100584 A2).

Antibodies may be produced by any of a number of techniques. Forexample, expression from host cells, wherein expression vector(s)encoding the heavy and light chains is (are) transfected into a hostcell by standard techniques. The various forms of the term“transfection” are intended to encompass a wide variety of techniquescommonly used for the introduction of exogenous DNA into a prokaryoticor eukaryotic host cell, e.g., electroporation, calcium-phosphateprecipitation, DEAE-dextran transfection and the like. Although it ispossible to express antibodies in either prokaryotic or eukaryotic hostcells, expression of antibodies in eukaryotic cells is preferable, andmost preferable in mammalian host cells, because such eukaryotic cells(and in particular mammalian cells) are more likely than prokaryoticcells to assemble and secrete a properly folded and immunologicallyactive antibody:

Preferred mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (includingdhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. AcadSci. USA 77:4216-4220, used with a DHER, selectable marker, e.g., asdescribed in R. T. Kaufman and P. A. Sharp (1982) Mol. Biol.159:601-621), NSO myeloma cells, COS cells and SP2 cells. Whenrecombinant expression vectors encoding antibody genes are introducedinto mammalian host cells, the antibodies are produced by culturing thehost cells for a period of time sufficient to allow for expression ofthe antibody in the host cells or, more preferably, secretion of theantibody into the culture medium in which the host cells are grown.Antibodies can be recovered from the culture medium using standardprotein purification methods.

Host cells can also be used to produce functional antibody fragments,such as Fab fragments or scFv molecules. It will be understood thatvariations on the above procedure are within the scope of the invention.For example, it may be desirable to transfect a host cell with DNAencoding functional fragments of either the light chain and/or the heavychain of an antibody of this invention. Recombinant DNA technology mayalso be used to remove some, or all, of the DNA encoding either or bothof the light and heavy chains that is not necessary for binding to theantigens of interest. The molecules expressed from such truncated DNAmolecules are also encompassed by the antibodies of the invention. Inaddition, bifunctional antibodies may be produced in which one heavy andone light chain are an antibody of the invention and the other heavy andlight chain are specific for an antigen other than the antigens ofinterest by crosslinking an antibody of the invention to a secondantibody by standard chemical crosslinking methods.

In a preferred system for recombinant expression of an antibody, orantigen binding portion thereof, a recombinant expression vectorencoding both the antibody heavy chain and the antibody light chain isintroduced into dhfr-CHO cells by calcium phosphate-mediatedtransfection. Within the recombinant expression vector, the antibodyheavy and light chain genes are each operatively linked to CMVenhancer/AdMLP promoter regulatory elements to drive high levels oftranscription of the genes. The recombinant expression vector alsocarries a DHFR gene, which allows for selection of CHO cells that havebeen transfected with the vector using methotrexateselection/amplification. The selected transformant host cells arecultured to allow for expression of the antibody heavy and light chainsand intact antibody is recovered from the culture medium. Standardmolecular biology techniques are used to prepare the recombinantexpression vector, transfect the host cells, select for transformants,culture the host cells and recover the antibody from the culture medium.Still further the invention provides a method of synthesizing arecombinant antibody of the invention by culturing a host cell in asuitable culture medium until a recombinant antibody is synthesized.Recombinant antibodies of the invention may be produced using nucleicacid molecules corresponding to the amino acid sequences disclosedherein The method can further comprise isolating the recombinantantibody from the culture medium.

The N- and C-termini of antibody polypeptide chains of the presentinvention may differ from the expected sequence clue to commonlyobserved post-translational modifications. For example, C-terminallysine residues are often missing from antibody heavy chains. Dick etal. (2008) Biotechnol. Bioeng. 100:1132. N-terminal glutamine residues,and to a lesser extent glutamate residues, are frequently converted topyroglutamate residues on both light and heavy chains of therapeuticantibodies. Dick et al. (2007) Biotechnol. Bioeng. 97:544; Liu et al.(2011) JBC 28611211; Liu et al. (2011) J. Biol. Chem. 286:11211.

III. Anti-B7-H3 Antibody Drug Conjugates (ADCs)

Anti-B7-H3 antibodies described herein may be conjugated to a drugmoiety to form an anti-B7-H3 Antibody Drug Conjugate (ADC).Antibody-drug conjugates (ADCs) may increase the therapeutic efficacy ofantibodies in treating disease, e.g., cancer, due to the ability of theADC to selectively deliver one or more drug moiety(s) to target tissues,such as a tumor-associated antigen, e.g., B7-H3 expressing tumors. Thus,in certain embodiments, the invention provides anti-B7-H3 ADCs fortherapeutic use, e.g., treatment of cancer.

Anti-B7-H3 ADCs of the invention comprise an anti-B7-H3 antibody, i.e.,an antibody that specifically binds to B7-H3, linked to one or more drugmoieties. The specificity of the ADC is defined by the specificity ofthe antibody, i.e., anti-B7-H3. In one embodiment, an anti-B7-H3antibody is linked to one or more cytotoxic drug(s) which is deliveredinternally to a transformed cancer cell expressing B7-H3.

Examples of drugs that may be used in the anti-B7-H3 ADC of theinvention are provided below, as are linkers that may be used toconjugate the antibody and the one or more drug(s). The terms “drug,”“agent,” and “drug moiety” are used interchangeably herein. The terms“linked” and “conjugated” are also used interchangeably herein andindicate that the antibody and moiety are covalently linked.

In some embodiments, the ADC has the following formula (formula I):

(D-L-LK_(m)Ab  (I)

wherein Ab is the antibody, e.g., anti-B7-H3 antibody huAb13v1,huAh3v2.5, or huAb3v2.6, and (L) is a linker, (D) is a drug, and LKrepresents a covalent linkage linking linker L to antibody Ab; and in isan integer ranging from 1 to 20. D is a drug moiety having, for example,cytostatic, cytotoxic, or otherwise therapeutic activity against atarget cell, e.g., a cell expressing B7-H3. In some embodiments, mranges from 1 to 8, 1 to 7, 1 to 6, 2 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to2, 1.5 to 8, 1.5 to 7, 1.5 to 6, 1.5 to 5, 1.5 to 4, 2 to 6, 1 to 5, 1to 4, 1 to 3, 1 to 2,or 2 to 4. The DAR of an ADC is equivalent to the“m” referred to in Formula I. In one embodiment, the ADC has a formulaof Ab-(LK-L-D)_(m), wherein Ab is an anti-B7-H3 antibody, e.g. huAb13v1,huAb3v2.5, or huAb3v2.6, L is a linker, D is a drug, e.g., a Bcl-xLinhibitor or an auristatin such as MMAF or MMAE, and m is 2 to 4(equivalent to a DAR of 2-4). Additional details regarding drugs (D ofFormula I) and linkers (L of Formula I) that may be used in the ADCs ofthe invention, as well as alternative ADC structures, are describedbelow.

III. A. Anti-B7-H3 ADCs: Bcl-xL Inhibitors, Linkers, Synthons, andMethods of Making Same

Dysregulated apoptotic pathways have also been implicated in thepathology of cancer. The implication that down-regulated apoptosis (andmore particularly the Bcl-2 family of proteins) is involved in the onsetof cancerous malignancy has revealed a novel way of targeting this stillelusive disease. Research has shown, for example, the anti-apoptoticproteins, Bel 2 and Bcl-xl, are over-expressed in many cancer celltypes. See, Zhang, 2002, Nature Reviews/Drug Discovery 1:101 Kirkin etal., 2004, Biochimica Biophysica Acta 1644:229-249; and Amundson et al.,2000, Cancer Research 60:6101-6110. The effect of this deregulation isthe survival of altered cells which would otherwise have undergoneapoptosis in normal conditions. The repetition of these defectsassociated with unregulated proliferation is thought to be the startingpoint of cancerous evolution.

Aspects of the disclosure concern anti-hB7-H3 ADCs comprising ananti-hB7-H3 antibody conjugated to a drug via a linker, wherein the drugis a Bcl-xL inhibitor. In specific embodiments, the ADCs are compoundsaccording to structural formula (I) below; or a pharmaceuticallyacceptable salt thereof, wherein Ab represents the anti-hB7-H3 antibody,D represents a Bcl-xL inhibitor drug (i.e., a compound of formula (IIa),(IIb), (IIc), or (IId) as shown below), L represents a linker, LKrepresents a covalent linkage linking the linker (L) to the anti-hB7-H3antibody (Ab) and m represents the number of D-L-LK units linked to theantibody, which is an integer ranging from 1 to 20. In certainembodiments, m is 2, 3 or 4. In some embodiments, in ranges from 1 to 8,1 to 7, 1 to 6, 2 to 6, 1 to 5, 1 to 4, 2 to 4, 1 to 3, 1 to 2, or is 1.

(D-L-LK_(m)Ab  (I)

Specific embodiments of various Bcl-xL inhibitors per se, and variousBcl-xL inhibitors (D), linkers (L) and anti-B7-H3 antibodies (Ab) thatcan comprise the ADCs described herein, as well as the number of Bcl-xLinhibitors linked to the ADCs, are described in more detail below.

Examples of Bcl-xL inhibitors that may be used in the anti-B7-H3 ADC ofthe invention are provided below, as are linkers that may be used toconjugate the antibody and the one or more Bcl-xL inhibitor(s). Theterms “linked” and “conjugated” are also used interchangeably herein andindicate that the antibody and moiety are covalently linked.

IIA.1. Bcl-xL Inhibitors

One aspect of the instant disclosure concerns Bcl-xL inhibitors thathave low cell permeability. The compounds are generally heterocyclic innature and include one or more solubilizing groups that impart thecompounds with high water solubility and low cell permeability. Thesolubilizing groups are generally groups that are capable of hydrogenbonding, forming dipole-dipole interactions, and/or that include apolyethylene glycol polymer containing from 1 to 30 units, one or morepolyols, one or more salts, or one or more groups that are charged atphysiological pH.

The Bcl-xL inhibitors may be used as compounds or salts per se in thevarious methods described herein, or may be included as a component partof an ADC.

Specific embodiments of Bcl-xL inhibitors that may be used inunconjugated form, or that 2c may be included as part of an ADC includecompounds according to structural formulae (Ha), (IIb), (IIc), or (IM).In the present invention, when the Bcl-xL inhibitors are included aspart of an ADC, # shown in structural formula (IIa), (IIb), (IIc), or(IId) below represents a point of attachment to a linker, whichindicates that they are represented in a monoradical form.

or a pharmaceutically acceptable salt thereof, wherein:

Ar¹ is selected from

and is optionally substituted with one or more substituentsindependently selected from halo, hydroxy, nitro, lower alkyl, lowerheteroalkyl, C₁₋₄ alkoxy, amino, cyano and halomethyl;

Ar² is selected from

and is optionally substituted with one or more substituentsindependently selected from halo, hydroxy, nitro, lower alkyl, lowerheteroalkyl, C₁₋₄alkoxy, amino, cyano and halomethyl, wherein theR¹²—Z^(2b)—, R′Z^(2b)—, #—N(R⁴)—R¹³—Z^(2b)—, or #—R′—Z^(2b)—substituents are attached to Ar² at any Ar² atom capable of beingsubstituted;

Z¹ is selected from N, CH, C-halo, C—CH₃ and C—CN;

Z^(2a) and Z^(2b) are each, independently from one another, selectedfrom a bond, NR⁶, CR^(6a)R^(6b), O, S, S(O), S(O)₂, —NR⁶C(O)—,—NR^(6a)C(O)NR^(6b)—, and —NR⁶C(O)O—.

R′ is a alkylene, heteroalkylene, cycloalkylene, heterocyclene, aryl orheteroaryl independently substituted at one or more carbon orheteroatoms with a solubilizing moiety containing a group selected froma polyol, a polyethylene glycol containing from 4 to 30 ethylene glycolunits, a salt, and a group that is charged at physiological pH andcombinations thereof, wherein where attached to is attached to R′ at anyR′ atom capable of being substituted;

R¹ is selected from hydrogen, methyl, halo, halomethyl, ethyl, andcyano;

R² is selected from hydrogen; methyl, halo, halomethyl and cyano;

R³ is selected from hydrogen, methyl, ethyl, halomethyl and haloethyl;

R⁴ is selected from hydrogen, lower alkyl and lower heteroalkyl or istaken together with an atom of R¹³ to form a cycloalkyl or heterocyclylring having between 3 and 7 ring atoms;

R⁶, R^(6a) and R^(6b) are each, independent from one another, selectedfront hydrogen, optionally substituted lower alkyl, optionallysubstituted lower heteroalkyl, optionally substituted cycloalkyl andoptionally substituted heterocyclyl, or are taken together with an atomfrom R⁴ and an atom from to form a cycloalkyl or heterocyclyl ringhaving between 3 and 7 ring atoms;

R^(11a) and R^(11b) are each, independently of one another, selectedfrom hydrogen, halo, methyl, ethyl, halomethyl, hydroxyl, methoxy, CN,and SCH₃;

R^(11a) is optionally R′ or is selected from hydrogen, halo, cyano,optionally substituted alkyl, optionally substituted heteroalkyl,optionally substituted heterocyclyl, and optionally substitutedcycloalkyl;

R¹³ is selected from optionally substituted C₁₋₈ alkylene, optionallysubstituted heteroalkylene, optionally substituted heterocyclene, andoptionally substituted cycloalkylene; and

# represents the point of attachment to a linker L.

One embodiment of Bcl-xL inhibitors that may be used in unconjugatedform, or that may be included as part of an ADC include compoundsaccording to structural formulae (IIa), (IIb), (IIc), or (IId):

or a pharmaceutically acceptable salt thereof, wherein:

Ar¹ is selected from

and is optionally substituted with one or more substituentsindependently selected from halo, hydroxy, nitro, lower alkyl, lowerheteroalkyl, C₁₋₄ alkoxy, amino, cyano and halomethyl;

Ar² is selected from

or an N-oxide thereof, and is optionally substituted with one or moresubstituents independently selected from halo, hydroxy, nitro, loweralkyl, lower heteroalkyl, C₁₋₄alkoxy, amino, cyano and halomethyl,wherein the R¹²—Z^(2b)—, R′—Z^(2b), #—N(R⁴)—R¹³—Z^(2b)—, #—R′—Z^(2b)— orsubstituents are attached to Ar² at any Ar² atom capable of beingsubstituted;

Z¹ is selected from N, CH, C-halo, C—CH₃ and C—CN;

Z^(2a) and Z^(2b) are each, independently from one another, selectedfrom a bond, NR⁶, CR^(6a)R^(6b), O, S, S(O), S(O)₂, —NR⁶C(O)—,—NR^(6a)C(O)NR^(6b)—, and NR⁶C(O)O—;

R′ is

wherein #, where attached to R′, is attached to R′ at any R′ atomcapable of being substituted;

X′ is selected at each occurrence from —N(R¹⁰)— —N(R¹⁰)C(O)—,—N(R¹⁰)S(O)₂—, —S(O)₂N(R¹⁰)—, and —O—;

n is selected from 0-3;

R¹⁰ is independently selected at each occurrence from hydrogen, loweralkyl, heterocycle, aminoalkyl, G-alkyl, and—(CH₂)₂—O—(CH₂)₂—O—(CH₂)₂—NH₂;

G at each occurrence is independently selected from a polyol, apolyethylene glycol with between 4 and 30 repeating units, a salt and amoiety that is charged at physiological pH;

SP^(a) is independently selected at each occurrence from oxygen,—S(O)₂N(H)—, —N(H)S(O)₂—, —N(H)C(O)—, —C(O)N(H)—, —N(H)—, arylene,heterocyclene, and optionally substituted methylene; wherein methyleneis optionally substituted with one or more of —NH(CH₂)₂G, NH₂,C₁₋₈alkyl, and carbonyl;

m² is selected from 0-12;

R¹ is selected from hydrogen, methyl, halo, halomethyl, ethyl, andcyano;

R² is selected from hydrogen, methyl, halo, halomethyl and cyano;

R³ is selected from hydrogen, methyl, ethyl, halomethyl and haloethyl;

R⁴ is selected from hydrogen, lower alkyl and lower heteroalkyl or istaken together with an atom of R¹³ to form a cycloalkyl or heterocyclylring having between 3 and 7 ring atoms;

R⁶, R^(6a) and R^(6b) are each, independent from one another, selectedfrom hydrogen, optionally substituted lower alkyl, optionallysubstituted lower heteroalkyl, optionally substituted cycloalkyl andoptionally substituted heterocyclyl, or are taken together with an atomfrom R⁴ and an atom from R¹³ to form a cycloalkyl or heterocyclyl ringhaving between 3 and 7 ring atoms;

R^(11a) and R^(11b) are each, independently of one another, selectedfrom hydrogen, halo, methyl, ethyl, halomethyl, hydroxyl, methoxy, CN,and SCH₃;

R¹² is optionally R′ or is selected from hydrogen, halo, cyano,optionally substituted alkyl, optionally substituted heteroalkyl,optionally substituted heterocyclyl, and optionally substitutedcycloalkyl;

R¹³ is selected from optionally substituted C₁₋₈ alkylene, optionallysubstituted heteroalkylene, optionally substituted heterocyclene, andoptionally substituted cycloalkylene; and

# represents the point of attachment to a linker L.

When a Bcl-xL, inhibitor of structural formulae (IIa)-(IId) is not acomponent of an ADC, # in formulae (IIa)-(IId) represents the point ofattachment to a hydrogen atom. When the Bcl-xL inhibitor is a componentof an ADC, # in formulae (IIa)-(IId) represents the point of attachmentto the linker. When a Bcl-xL inhibitor is a component of an ADC, the ADCmay comprise one or more Bcl-xL inhibitors, which may be the same ordifferent, but are typically the same.

In certain embodiments, R′ is a C₂-C₈ heteroalkylene substituted withone or more moieties containing a salt and/or a group that is charged atphysiological pH. The salt may be selected, for example, from the saltof a carboxylate, a sulfonate, a phosphonate, and an ammonium ion. Forexample, the salt may be the sodium or potassium salt of a carboxylate,sulfonate or phosphonate or the chloride salt of an ammonium ion. Thegroup that is charged at physiological pH may be any group that ischarged at a physiological pH, including, by way of example and notlimitation, a zwitterionic group. In certain embodiments a group that isa salt is a dipolar moiety such as, but not limited to, N-oxides ofamines including certain heterocyclyls such as, but not limited to,pyridine and quinoline. In specific embodiments the group that ischarged at physiological pH is selected independently at eachoccurrence, from carboxylate, sulfonate, phosphonate, and amine.

In certain embodiments, R′ is a C₂-C₈ heteroalkylene substituted withone or more moieties a containing polyethylene glycol or a polyol suchas a diol or a sugar moiety.

In certain embodiments, R′ may be substituted with groups in addition toa solubilizing moiety. For example, R′ may be substituted with one ormore of the same or different alkyl, heteroalkyl, cycloalkyl,heterocyclyl, aryl, heteroaryl, or halo groups.

In certain embodiments, R′ is represented by the formula:

or a pharmaceutically acceptable salt thereof, wherein:

X′ is selected at each occurrence from —N(R¹⁰)— and —O—;

n is selected from 1-3;

R¹⁰ is individually selected at each occurrence from hydrogen, alkyl,heterocycle, aminoalkyl, G-alkyl, heterocycle, and—(CH²)₂—O—(CH2)₂-O—(CH₂)₂—NH₂;

G at each occurrence is independently selected from a polyol, apolyethylene glycol with between 4 and 30 repeating unit (referred toherein as PEG4-30), a salt and a moiety that is charged at physiologicalpH;

SP^(a) is independently selected at each occurrence from oxygen,sulfonamide, arylene, heterocyclene, and optionally substitutedmethylene; wherein methylene is optionally substituted with one or moreof —NH(CH₂)₂G, amine and carbonyl; and

m² is selected from 0-6.

wherein there is at least one substitutable nitrogen in R′ that isattached to a linker or a hydrogen atom at a substitutable nitrogen atomof R′.

In certain embodiments, R′ is

X′ is selected at each occurrence from —N(R¹⁰)—, —N(R¹⁰)C(O)—,—N(R¹⁰)S(O)₂—, —S(O)₂N(R¹⁰)— and —O—;

n is selected from 0-3;

R¹⁰ is independently selected at each occurrence from hydrogen, alkyl,heterocycle, aminoalkyl, G-alkyl, heterocycle, and—(CH₂)₂—O—(CH₂)₂—O—(CH₂)₂—NH₂;

G at each occurrence is independently selected from a polyol, apolyethylene glycol with between 4 and 30 repeating units, a salt and amoiety that is charged at physiological pH;

SP^(a) is independently selected at each occurrence fromoxygen-S(O)₂N(H)—, —N(H)S(O)₂—, —N(H)C(O)—, —C(O)N(H)—, —N(H)—, arylene,heterocyclene, and optionally substituted methylene; wherein methyleneis optionally substituted with one or more of —NH(CH₂)₂G, amine, alkyl,and carbonyl;

m² is selected from 0-12, and

#, where attached to R′, is attached to R′ at any R′ atom capable ofbeing substituted.

In certain embodiments, G at each occurrence is a salt or a moiety thatis charged at physiological pH.

In certain embodiments, G at each occurrence is a salt of a carboxylate,a sulfonate, a phosphonate, or ammonium.

In certain embodiments, G at each occurrence is a moiety that is chargedat physiological pH selected from the group consisting of carboxylate, asulfonate, a phosphonate, and an amine.

In certain embodiments, G at each occurrence is a moiety containing apolyethylene glycol with between 4 and 30 repeating units, or a polyol.

In certain embodiments, the polyol is a sugar.

In certain embodiments, R′ of formula (IIa) or (I Id) includes at leastone substitutable nitrogen suitable for attachment to a linker.

In certain embodiments, G is selected independently at each occurrencefrom:

wherein M is hydrogen or a positively charged counterion. In certainembodiments, M is Na⁺, K⁺ or Li⁺. In certain embodiments, M is hydrogen.In particular embodiments, G is SO₃H.

In certain embodiments, G is selected independently at each occurrencefrom:

wherein M is hydrogen or a positively charged counterion. In certainembodiments, M is hydrogen. In particular embodiments, G is SO₃H.

In certain embodiments, R′ is selected from:

or a salt thereof. When Bcl-xL inhibitors of this embodiment areincluded in an ADC, the linker of the ADC is linked to the nitrogen atomof an available primary or secondary amine group.

In certain embodiments, R′ is selected from:

or a salt thereof. When Bcl-xL inhibitors of this embodiment areincluded in an ADC, the linker of the ADC is linked to the nitrogen atomof an available primary or secondary amine group.

In certain embodiments, R′ is selected from

wherein # represents either a hydrogen atom in the Bcl-xL inhibitor drugof the ADCs of formula (IIb) or (IIc) or the point of attachment in theBcl-xL inhibitor drug of the ADCs of formula (IIa) or (IId) to a linkerL.

In certain embodiments, Ar¹ of formulae (IIa)-(IId) is selected from

In certain embodiments, Ar¹ of formulae (IIa)-(IId) is selected from

and is optionally substituted with one or more substituentsindependently selected from halo, cyano, methyl, and halomethyl. Inparticular embodiments, Ar¹ is

In certain embodiments, AR² is

optionally substituted with one or more substituents, wherein theR¹²—Z^(2b)—, #—N(R⁴)—R¹³—Z^(2b)—, or #—R′—Z^(2b)— substituents areattached to Ar² at any Ar² atom capable of being substituted.

In certain embodiments, Ar² is selected from:

and is optionally substituted with one or more substituents, wherein theR¹²—Z^(2b)—, R′—Z^(2b)—, #—N(R⁴)—R¹³—Z^(2b)—, or #—R′—Z^(2b)—substituents are attached to Ar² at any Ar² atom capable of beingsubstituted. In certain embodiments, Ar² is selected from:

and is optionally substituted with one or more substituents, wherein theR¹²—Z^(2b)—, R′—Z^(b)—, #—N(R⁴)—R¹³—Z^(2b)— or #—R′Z^(2b)— substituentsare attached to Ar² at any Ar² atom capable of being substituted. Incertain embodiments, Ar² is substituted with one or more solubilizinggroup. In certain embodiments, the each solubilizing group is,independently of the others, selected from a moiety containing a polyol,a polyethylene glycol with between 4 and 30 repeating units, a salt, ora moiety that is charged at physiological pH.

In certain embodiments, Z¹ of formulae (IIa)-(IId) is N.

In certain embodiments, Z^(2a) of formulae (IIa)-(IId) is O. In certainembodiments, Z^(2a) of formulae (IIa)-(IId) is CR^(6a)R^(6b). In certainembodiments, Z^(2a) of formulae (IIa)-(IId) is S. In certainembodiments, Z^(2a) of formulae (IIa)-(IId) is NR⁶C(O)—. In particularembodiments, R⁶ is hydrogen.

In certain embodiments, Z^(2b) of formulae (IIa)-(IId) is O. In certainembodiments, Z^(2b) of formulae (IIa)-(IId) is NH or CH₂.

In certain embodiments, R¹ of formulae (IIa)-(IId) is selected frommethyl and chloro.

In certain embodiments, R² of formulae (IIa)-(IId) is selected fromhydrogen and methyl. In particular embodiments, R² is hydrogen.

In certain embodiments the Bcl-xL inhibitor is a compound of formula(IIa). In certain embodiments in which the Bcl-xL inhibitor is acompound of formula (IIa), the compound has the structural formula(IIa.1),

or salts thereof wherein:

Ar¹, Ar², Z¹, Z^(2a), Z^(zb), R¹, R², R^(11a), R^(11b), R^(12′) G and #are defined as above;

Y is optionally substituted. C₁-C₈ alkylene;

r is 0 or 1; and

s is 1, 2 or 3.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIa.1), r is 0 and s is 1.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIa.1), r is 0 and s is 2.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIa.1), r is 1 and s is 2.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIa.1), Z² is selected from O, NH, CH₂ and S. In particularembodiments, Z^(2a) is O. In certain embodiments, Z^(2a) of formula(IIa.1) is —CR^(6a)R^(6b)—. In certain embodiments, Z^(2a) of formula(IIa.1) is CH₂. In certain embodiments, Z^(2a) of formula (IIa.1) is S.In certain embodiments, Z² of formula (IIa.1) is —NR⁶C(O)—.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIa.1), Y is selected from ethylene, propylene and butylene. Inparticular embodiments, Y is selected from ethylene and propylene.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIa.1), G is selected from

wherein M is hydrogen or a positively charged counterion. In particularembodiments, G is

In particular embodiments. G is SO₃H.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIa.1), Ar² is selected from

wherein the R¹²—Z^(2b)— substituent is attached to Ar² at any Ar² atomcapable of being substituted.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIa.1), Ar² is selected from

wherein the R¹²—Z^(2b)-substituent is attached to Ar² at any Ar² atomcapable of being substituted.

In particular embodiments in which the Bcl-xL inhibitor is a compound offormula (IIa.1), Ar² is

In particular embodiments in which the Bcl-xL inhibitor is a compound offormula (IIa.1). Ar² is

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIa.1), Z^(2b)—R¹² is selected from H, F, CN, OCH_, OH, NH₂,OCH₂CH₂OCH₃, N(CH₃)C(═O)CH₃, CH₂N(CH₃)C(═O)CH₃SCH₃, C(═O)N(CH₃)₂ andOCH₂CH₂N(CH₃)(C(═O)CH₃). In particular embodiments, Z^(2b)—R¹² isselected from H, F and CN. In particular embodiments, Z^(2b)—R¹² is H.

In embodiments where Z^(2b)-R¹² is substituted with hydroxyl (OH), theoxygen can serve as the point of attachment to a linking group (SeeSection 4.4.1.1).

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIa.1), Ar¹ is

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIa.1), the group

bonded to the adamantane ring is selected from:

In certain embodiments, a compound of formula (IIa.1) may be convertedinto the compound of formula IIa.1.1, wherein n is selected from 1-3:

In certain embodiments, the compound of formula IIa.1.1 can be convertedinto a compound of formula IIa.1.2, wherein L represents a linker and LKrepresents a linkage formed between a reactive functional group onlinker L and a complementary functional group on antibody.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIa), the compound has the structural formula (IIa.2),

or salts thereof, wherein:

Ar¹,Ar², Z¹, Z^(2a), Z^(2b), R¹, R², R^(11a), R^(11b), R¹² and # aredefined as above;

U is selected from N, O and CH, with the proviso that when U is O, thenV^(a) and R^(21a) are absent;

R²⁰ is selected from H and C₁-C₄ alkyl;

R^(21a) and R^(21b) are each, independently from one another, absent orselected from H, alkyl and G, where G is selected from a polyol,PEG4-30, a salt and a moiety that is charged at physiological pH;

V^(a) and V^(b) are each, independently from one another, absent orselected from a bond, and an optionally substituted alkylene;

R²⁰ is selected from H and C₁-C₄ alkyl; and

s is 1, 2 or 3.

In certain embodiments in which the Bcl-xL inhibitor is a compound offoemula (IIa.2), s is 2.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIa.2), Z² is selected from O, NH, CH₂ and S. In particularembodiments, Z^(2a) is O. In certain embodiments, Z^(2a) of formula(II.a.2) is CR^(6a)R^(6b). In certain embodiments, Z^(2a) of formula(IIa.2) is CH₂. In certain embodiments, Z^(2a) of formula (IIa.2) is S.In certain embodiments, Z^(2a) of formula —NR⁶C(O)—.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIa.2), U is selected from N and O. In particular embodiments,U is O.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIa.2), V^(a) is a bond, R^(21a) is a C₁-C₄ alkyl group. V^(b)is selected, from methylene and ethylene and R^(21b) is G. In particularembodiments, V^(a) is a bond, R^(21a) is a methyl group and V^(b) isselected from methylene and ethylene and R^(21b) is G.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIa.2), V^(a) is selected from methylene and ethylene. R^(21a)is G, V^(b) is selected from methylene and ethylene and R^(21b) is G. Inparticular embodiments. V^(a) is ethylene, R²¹ is G, V^(b) is selectedfrom methylene and ethylene and R^(21b) is G.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIa.2), G is selected from

wherein M is hydrogen or a positively charged counterion. In particularembodiments, G is

In particular embodiments, G is SO₃H.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIa.2), R²⁰ is selected from hydrogen and a methyl group.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIa.2), Ar² is selected from

wherein the R¹²—Z^(2b)— substituent is attached to Ar² at any Ar² atomcapable of being substituted.

In certain embodiments in which the Bcl-xL, inhibitor is a compound offormula (IIa.2), Ar² is selected from

wherein the

R¹³—Z^(2b)— substituent is attached to Ar² at any Ar² atom capable ofbeing substituted.

In particular embodiments in which the Bcl-xL inhibitor is a compound offormula (IIa.2), Ar² is

wherein the R¹²—Z^(2b)— substituent is attached to Ar² at any Ar² atomcapable of being substituted.

In certain embodiments in which the Bcl-xL, inhibitor is a compound offormula (IIa.2), Z^(2b)—R¹² is selected from H, F, CN, OCH₃, OH, NH₂,OCH₂CH₂OCH₃, N(CH₃)C(═O)CH₃, CH₂N(CH₃)C(═O)CH₃SCH₃, C(═O)N(CH₃)₂ andOCH₂CH₂N(CH₃)(C(═O)CH₃). In particular embodiments, Z^(2b)—R¹² isselected from H, F and CN. In particular embodiments, Z^(12b)-R¹² is H.In certain embodiments in which the Bcl-xL, inhibitor is a compound offormula (IIa.2), Ar¹ is

In particular embodiments in which the Bcl-xL, inhibitor is a compoundof formula (IIa.2), Ar² is

wherein the R¹²—Z^(2b)— substituent is attached to Ar² at any Ar² atomcapable of being substituted.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIa), the compound has the structural formula (IIa.3),

or salts thereof, wherein:

Ar¹, Ar², Z¹, Z^(2a), Z^(2b), R¹, R², R^(11a), R¹² and # are defined asabove;

R^(b) is selected from H C₁-C₄ alkyl and J^(b)-G or is optionally takentogether with an atom of T to form a ring having between 3 and 7 atoms;

J^(a) and J^(b) are each, independently from one another, selected fromoptionally substituted C₁-C₈ alkylene and optionally substitutedphenylene;

T is selected from optionally substituted C₁-C₈ alkylene,CH₂CH₂OCH₂CH₂OCH₂CH₂, CH₂CH₂OCH₂CH₂OCH₂CH₂OCH₂ and a polyethylene glycolcontaining from 4 to 10 ethylene glycol units;

G is selected from a polyol, PEG4-30, a salt and a moiety that ischarged at physiological pH; and

s is 1, 2 or 3.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIa.3), s is 1.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIa.3), s is 2.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIa.3), Z^(2a) is selected from O, CH₂ and S. In particularembodiments, Z^(2a) is O. In certain embodiments, Z^(2a) of formula(IIa.3) is CR^(6a)R^(6b). In certain embodiments, Z^(2a) of formula(IIa.3) is CH₂. In certain embodiments, Z^(2a) of formula (IIa.3) is S.In certain embodiments, Z^(2a) of formula (IIa.3) is —NR⁶C(O)—.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIa.3), J^(a) is selected from methylene and ethylene and R^(b)is J^(b)-G, wherein J^(b) is methylene or ethylene. In some suchembodiments, T is ethylene. In other such embodiments, T isCH₂CH₂OCH₂CH₂OCH₂CH₂. In other such embodiments, T is a polyethyleneglycol containing from 4 to 10 ethylene glycol units.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIa.3), J^(a) is selected from methylene and ethylene and R^(b)is taken together with an atom of T to form a ring having 4-6 ringatoms.

In certain embodiments in which the Bcl-xL, inhibitor is a compound offormula (IIa.3), Ja is selected from methylene and ethylene and R^(h) isH or alkyl. In some such embodiments, T is ethylene. In other suchembodiments, T is CH₂CH₂OCH₂CH₂OCH₂CH₂.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula G is selected from

wherein M is hydrogen or a positively charged counterion. In particularembodiments, G is

In particular embodiments. G is SO₃H.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIa.3), R²⁰ is selected from hydrogen and a methyl group.

In certain embodiments in which the Bcl-xL inhibitor is a compound ofform Ar² is selected from

wherein the R¹²—Z^(2b)— substituent is attached to Ar² at any Ar² atomcapable of being substituted.

In particular embodiments in which the Bcl-xL inhibitor is a compound offormula (IIa.3), is

wherein the R¹²—Z^(2b)— substituent is attached to Ar² at any Ar² atomcapable of being substituted. In certain embodiments in which the Bcl-xLinhibitor is a compound of formula (IIa.3), Ar² is selected from

wherein the R¹²—Z^(2b)-substituent is attached to Ar² at any Ar² atomcapable of being substituted. In particular embodiments in which theBcl-xL inhibitor is a compound of formula (IIa.3), Ar² is

wherein the R¹²—Z^(2b)-substituent is attached to Ar² at any Ar² atomcapable of being substituted.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIa.3), Z^(2b)—R¹² is selected from H F, CN, OCH₃, OH, NH₂,OCH₂CH₂OCH₃, N(CH₃)C(═O)CH₃, CH₂N(CH₃)C(═O)CH₃SCH₃, C(═O)N(CH₃)₂ andOCH₂CH₂N(CH₃)(C(═O)CH₃). In particular embodiments, Z^(2b)—R¹² isselected from 14, F and CN. In particular embodiments, Z^(2b)—R¹² is H.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIa.3), Ar¹ is

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIa.3), the group

is selected from:

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIa.3), the group

is selected from:

In certain embodiments the Bcl-xL inhibitor is a compound of formula(IIb). In certain embodiments in which the Bcl-xL inhibitor is acompound of formula (IIb), the compound has the structural formula(IIb.1),

or salts thereof, wherein:

Ar¹, Ar², Z¹, Z^(2a), Z^(2b), R¹, R², R⁴, R^(11a), R^(11b) and # aredefined as above;

Y is optionally substituted C₁-C₈ alkylene;

G is selected from a polyol, PEG4-30, a salt and a moiety that ischarged at physiological pH;

r is 0 or 1; and

s is 1, 2 or 3.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIb.1), s is 1. In certain embodiments in which the Bcl-xLinhibitor is a compound of formula (IIb.1), s is 2. In certainembodiments in which the Bcl-xL inhibitor is a compound of formula(IIb.1), s is 3.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIb.1), Z^(2a) is selected from O, CH₂, NH and S. In particularembodiments, Z^(2a) is O. In certain embodiments, Z^(2a) of formula(IIb.1) is CR^(6a)R^(6b). In certain embodiments, Z^(2a) of formula(IIb.1) is CH₂. In certain embodiments, Z^(2a) of formula (IIb.1) is S.In certain embodiments, Z^(2a) of formula (IIb.1) is —NR⁶C(O)—.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIb.1), Z^(2b) is selected from O, CH₂, NH, NCH₃ and S. Inparticular embodiments, Z^(2b) is O. In particular embodiments, Z^(2b)is NH. In particular embodiments, Z^(2b) is NCH₃.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIb.1), Y is ethylene and r is 0.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIb.1), Y is ethylene and r is 1.

In certain embodiments in which the Bcl-xL, inhibitor is a compound offormula (IIb.1), R⁴ is H or methyl. In particular embodiments, R⁴ ismethyl. In other embodiments, R⁴ is H.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIb.1), R⁴ is taken together with an atom of Y to form a ringhaving 4-6 ring atoms. In particular embodiments, the ring is acyclobutane ring. In other embodiments, the ring is a piperazine ring.In other embodiments, the ring is a morpholine ring.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIb.1), G is selected from

wherein M is hydrogen or a positively charged counterion. In particularembodiments, G is

In other embodiments, U is SO₃H. In particular embodiments, G is NH₂. Inother embodiments, G is PO₃H₂. In particular embodiments, G is NH₂. Inparticular embodiments, G is C(O)OH. In particular embodiments, G ispolyol.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIb.1), Ar² is selected from

wherein the G-(CH₂)_(s)—Z^(2b)— substituent is attached to Ar² at anyAr² atom capable of being substituted.

In particular embodiments in which the Bcl-xL inhibitor is a compound offormula (IIb.1), Ar² is

wherein the G-(CH₂)_(s)—Z^(2b)— substituent is attached to Ar² at anyAr² atom capable of being substituted. In certain embodiments in whichthe Bcl-xL inhibitor is a compound of formula (IIb.1). Ar² is selectedfrom

wherein the G-(CH₂)_(a)—Z^(2b)— substituent is attached to Ar² at anyAr² atom capable of being substituted. In particular embodiments inwhich the Bcl-xL inhibitor is a compound of formula (IIb.1), Ar² is

wherein the G-(CH₂)_(s)—Z^(2b)— substituent is attached to Ar⁷ at anyAr² atom capable of being substituted.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIb.1), Ar¹ is

In certain embodiments in which the Bcl-xL, inhibitor is a compound offormula (IIb.1), the group

is selected from:

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIb.1), the group

is selected from:

In certain embodiments in which the Bcl-xL, inhibitor is a compound offor (IIb.1), the group

is selected from:

In certain embodiments the Bcl-xL inhibitor is a compound of formula(IIc). In certain embodiments in which the Bcl-xL inhibitor is acompound of formula (IIc), the compound has the structural formula(IIc.1)

or salts thereof, wherein:

Ar¹, Ar², Z¹, Z^(2a), Z^(2b), R¹, R², R⁴, R^(11a), R^(11b) and # aredefined as above;

Y^(a) is optionally substituted C₁-C₈ alkylene;

Y^(b) is optionally substituted C₁-C₈ alkylene;

R²³ is selected from H and C₁-C₄ alkyl; and

G is selected from a polyol, PEG4-30, a salt and a moiety that ischarged at physiological pH;

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIc.1), Z^(2a) is selected from O, CH₂, NH and S. In particularembodiments, Z^(2a) is O. In certain embodiments, Z^(2a) of formula(IIc.1) is CR^(6a)R^(6b). In certain embodiments. Z^(2a) of formula(IIc.1) is S. In certain embodiments, Z^(2a) of formula (IIc.1) is—NR⁶C(O)—.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIc.1), Z^(2b) is selected from O, CH₂, NH, NCH₃ and S. Inparticular embodiments, Z^(2b) is O. In particular embodiments, Z^(2a)is NFL In particular embodiments, Z^(2a) is NCH₃.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIc.1), Z^(2b) is a bond. In some such embodiments Y^(a) ismethylene or ethylene.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIc.1), is O. In some such embodiments Y^(a) is methylene,ethylene, or propylene.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIc.1), Z^(2b) is NR⁶, where R⁶ is defined as above. In somesuch embodiments, R⁶ is taken together with an atom from Y^(a) to form acycloalkyl or heterocyclyl ring having between 3 and 7 ring atoms. Insome such embodiments, the ring has 5 atoms.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIc.1), Y^(a) is ethylene.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIc.1), Y^(a) is methylene.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIc.1), Y^(a) is propylene.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIc.1), R⁴ is H. H or methyl. In particular embodiments, R⁴ isH.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIc.1), Y^(b) is ethylene or propylene. In particularembodiments, Y^(b) is ethylene.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIc.1), R²³ is methyl.

In certain embodiments in which c Bcl-xL inhibitor is a compound offormula (IIc.1), R²³ is H.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIc.1), G is selected from

wherein M is hydrogen or a positively charged counterion. In particularembodiments, G is

In particular embodiments, G is SO₃H

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIc.1), Ar² is selected from

wherein the #—N(R⁴)—Y^(a)—Z^(2b)-substituent is attached to Ar² at anyAr² atom capable of being substituted.

In particular embodiments in Which the Bcl-xL inhibitor is a compound offormula (IIc.1), Ar² is

wherein the #—N(R⁴)—Y^(a)—Z^(2b)— substituent is attached to Ar² at anyAr² atom capable of being substituted. In certain embodiments in whichthe Bcl-xL inhibitor is a compound of formula (IIc.1), Ar² is selectedfrom

wherein the #—N(R⁴)—Y^(a)—Z^(2b)— substituent is attached to Ar² at anyAr² atom capable of being substituted. In particular embodiments inwhich the Bcl-xL inhibitor is a compound of formula (IIc.1), Ar² is

wherein the #—N(R⁴)—Y^(a)—Z^(2b)— substituent is attached to Ar² at anyAr² atom capable of being substituted.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIc.1), Ar¹ is

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIc.1),the group

is selected from:

In other embodiments in which the Bcl-xL inhibitor is a compound offormula (IIc.1), the group

is selected from:

In certain embodiments in which the Bcl-xL inhibitor is a compound offor (IIc), the compound has the structural formula (IIc.2),

or salts thereof wherein:

Ar¹, Ar², Z¹, Z^(2a), Z^(2b), R¹, R², R⁴, R^(11a), R^(11b) and # aredefined as above;

Y^(a) is optionally substituted C₁-C₈ alkylene;

Y^(b) is optionally substituted C₁-C₈ alkylene;

Y^(c) is optionally substituted C₁-C₈ alkylene;

R²³ is selected from H and C₁-C₄ alkyl;

R²⁵ is Y^(b)-G or is taken together with an atom of Y^(c) to form a ringhaving 4-6 ring atoms; and

G is selected from a polyol, PEG4-30, a salt and a moiety that ischarged at physiological pH.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIc.2), Z^(2a) is selected from O, CH₂, NH and S. In particularembodiments, Z^(2a) is O. In certain embodiments, Z^(2a) of formula(IIc.2) is CR^(6a)R^(6b). In certain embodiments, Z² of formula (IIc.2)is S. In certain embodiments, Z^(2a) of formula (IIc.2) is —NR⁶C(O)—.Incertain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIc.2), Z^(2b) is selected from O, CH₂, NH, NCH₃ and S. Inparticular embodiments, Z^(2b) is O. In particular embodiments, Z² isNH. In particular embodiments, Z^(7b) is NCH₃.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIc.2), Z^(2b) is a bond. In some such embodiments Y^(a) ismethylene or ethylene.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIc.2), Z^(2b) is NR⁶, where R⁶ is defined as above. In somesuch embodiments, R^(b) is taken together with an atom from Y^(a) toform a cycloalkyl or heterocyclyl ring having between 3 and 7 ringatoms. In some such embodiments, the ring has 5 atoms.

In certain embodiments in which the Bcl-xL, inhibitor is a compound offormula (IIc.2), r is ethylene.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIc.2), Y^(a) is methylene.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIc.2), R⁴ is H or methyl.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIc.2), Y^(b) is ethylene or propylene. In particularembodiments, Y^(b) is ethylene.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIc.2), Y^(c) is ethylene or propylene. In particularembodiments, Y^(b) is ethylene.

In certain embodiments in which the Bcl-xL, inhibitor is a compound offormula (IIc.2), R²⁵ is taken together with an atom of Y^(c) to form aring having 4 or 5 ring atoms.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIc.2), R²³ is methyl.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIc.2), G is selected from

wherein M is hydrogen or a positively charged counterion. In particularembodiments, G is

In particular embodiments, G is SO₃H.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIc.2), Ar² is selected from

wherein the #—N(R⁴)—Y^(a)—Z^(2b)— substituent is attached to Ar² at anyAr² atom capable of being substituted.

In particular embodiments in which the Bcl-xL inhibitor is a compound offormula (IIc.2), Ar² is

wherein the #—N(R⁴)—Y^(a)—Z^(2b)— substituent is attached to Ar² at anyAr² atom capable of being substituted. In certain embodiments in whichthe Bcl-xL inhibitor is a compound of formula (IIc.2), Ar² is selectedfrom

wherein the #—N(R⁴)—Y^(a)—Z^(2b)— substituent is attached to Ar² at anyAr² atom capable of being substituted. In particular embodiments inwhich the Bcl-xL inhibitor is a compound of formula (IIc.2), Ar² is

wherein the #—N(R⁴)—Y^(a)—Z^(2b)— substituent is attached to Ar² at anyAr² atom capable of being substituted.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIc.2), Ar¹ is

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IIc.2), the group

is selected from:

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IId), the compound has the structural formula (IId.1),

or salts thereof, wherein:

Ar¹, Ar², Z¹, Z^(2a), Z^(2b), R¹, R², R^(11a), R^(11b) and # are definedas above;

Y^(a) is optionally substituted alkylene;

Y^(b) is optionally substituted alkylene;

R²³ is selected from H and C₁-C₄ alkyl;

G^(a) is selected from a polyol, PEG4-30, a salt and a moiety that ischarged at physiological pH;

G^(b) is selected from a polyol, PEG4-30, a salt and a moiety that ischarged at physiological pH;

In certain embodiments in which the Bcl-xL, inhibitor is a compound offormula (IId.1), s is 1.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IId.1), s is 2.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IId.1). Z^(2a) is selected from O, NH, CH₂ and S. In particularembodiments, Z^(2a) is O. In certain embodiments, Z^(2a) of formula(IId.1) is CR^(6a)R^(6b). In certain embodiments, Z^(2a) of formula) isS. In certain embodiments, Z^(2a) of formula (IId.1) is —NR⁶C(O)—.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IId.1), Z^(2b) is selected from O, NH, CH₂ and S. In particularembodiments, Z^(2b) is O.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IId.1), selected from ethylene, propylene and butylene. Inparticular embodiments, Y^(a) is ethylene.

In certain embodiments in which the Bcl-xL, inhibitor is a compound offormula (IId.1), Y^(a) is selected from ethylene, propylene andbutylene. In particular embodiments, Y is ethylene.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IId.1). G^(a) is selected from

wherein M is hydrogen or a positively charged counterion. In particularembodiments, G^(a) is

In particular embodiments, G^(a) is SO₃H. In particular embodiments,G^(a) is CO₂H.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IId.1), G^(b) is selected from

wherein M is hydrogen or a positively charged counterion. In particularembodiments, G^(b) is

In particular embodiments, G^(b) is SO₃H. In particular embodiments,G^(b) is CO₂H.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IId.1), R²³ is methyl.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IId.1), Ar² is selected from

wherein the G^(a)-Y^(a)—N(#)—(CH₂)_(s)—Z^(2b)— substituent is attachedto Ar² at any Ar² atom capable of being substituted.

In particular embodiments in which the Bcl-xL inhibitor is a compound offormula (IId.1), Ar² is

wherein the G^(a)-Y^(a)—N(#)—(CH₂)_(s)—Z^(2b)— substituent is attachedto Ar² at any Ar² atom capable of being substituted. In certainembodiments in which the Bcl-xL inhibitor is a compound of formula(IId.1), Ar² is selected from

wherein the G^(a)-Y^(a)—N(#)—(CH₂)_(s)—Z^(2b)— substituent is attachedto Ar² at any Ar² atom capable of being substituted. In particularembodiments in which the Bcl-xL inhibitor is a compound of formula(IId.1), Ar² is

wherein the G^(a)—Y^(a)—N(#)—(CH₂)_(s)—Z^(2b)— substituent is attachedto Ar² at any Ar² atom capable of being substituted.

In certain embodiments in which the Bcl-xL inhibitor is a compound offormula (IId.1), Ar¹ is

In certain embodiments, R^(11a) and R^(11b) of formulae (IIa)-(IId) arethe same. In a particular embodiment, R^(11a) and R^(11b) are eachmethyl.

In certain embodiments, the compounds of formulae (IIa)-(IId) includeone of the following cores (C.1)-(C.21):

Exemplary Bcl-xL inhibitors according to structural formulae (IIa)-(IId)that may be used in the methods described herein in unconjugated formand/or included in the ADCs described herein include the followingcompounds, and/or salts thereof:

App Ex. No. Bcl-xL Inhibitor Cmpd No 1.1 W2.01 1.2 W2.02 1.3 W2.03 1.5W2.05 1.6 W2.06 1.7 W2.07 1.8 W2.08 1.9 W2.09 1.10 W2.10 1.11 W2.11 1.12W2.12 1.13 W2.13 1.14 W2.14 1.15 W2.15 1.16 W2.16 1.17 W2.17 1.18 W2.181.19 W2.19 1.20 W2.20 1.21 W2.21 1.22 W2.22 1.23 W2.23 1.24 W2.24 1.25W2.25 1.26 W2.26 1.27 W2.27 1.28 W2.28 1.29 W2.29 1.30 W2.30 1.31 W2.311.32 W2.32 1.33 W2.33 1.34 W2.34 1.35 W2.35 1.36 W2.36 1.37 W2.37 1.38W2.38 1.39 W2.39 1.40 W2.40 1.41 W2.41 1.42 W2.42 1.43 W2.43 1.44 W2.441.45 W2.45 1.46 W2.46 1.47 W2.47 1.48 W2.48 1.49 W2.49 1.50 W2.50 1.51W2.51 1.52 W2.52 1.53 W2.53 1.54 W2.54 1.55 W2.55 1.56 W2.56 1.57 W2.571.58 W2.58 1.59 W2.59 1.60 W2.60 1.61 W2.61 1.62 W2.62 1.63 W2.63 1.64W2.64 1.65 W2.65 1.66 W2.66 1.67 W2.67 1.68 W2.68 1.69 W2.69 1.70 W2.701.71 W2.71 1.72 W2.72 1.73 W2.73 1.74 W2.74 1.75 W2.75 1.76 W2.76 1.77W2.77 1.78 W2.78 1.79 W2.79 1.80 W2.80 1.81 W2.81 1.82 W2.82 1.83 W2.831.84 W2.84 1.85 W2.85 1.86 W2.86 1.87 W2.87 1.88 W2.88 1.89 W2.89 1.90W2.90 1.91 W2.91

Notably, when the Bcl-xL inhibitor of the present application is inconjugated form, the hydrogen corresponding to the position ofstructural formulae (IIa)-(IId) is not present, forming a monoradical.For example, compound W2.01 (Example 1.1) is6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-[1-({3-[2-({2-[2-(carboxymethoxy)ethoxy]ethyl}aminoethoxy]-5,7-dimethyltricyclo[3.3.1,13,7]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylicacid.

When it is in unconjugated form, it has the following structure:

When the same compound is included in the ADCs as shown in structuralformula (IIa) or (IIb), the hydrogen corresponding to the # position isnot present, forming a monoradical.

In certain embodiments, the Bcl-xL inhibitors according to structuralformulae (IIa)-(IId) are selected from the group consisting of W2.01,W2.02, W2.03, W2.04, W2.05, W2.06, W2.07, W2.08, W2.09, W2.10, W2.11,W2,12, W2.13, W2.14, W2.15, W2A6, W2,17, W2.18, W2.19, W2.20, W2.21,W2,22, W2.23, W2.24, W2.25, W2,26, W2.27, W2.28, W2.29, W2.30, W2,31,W2.32, W2.33, W234, W2.35, W2.36, W2.37, W2.38, W2.39, W2.40, W2.41,W2.42, W2.43, W2.44, W2.45, W2.46, W2.47, W2,48, W2.49, W2.50, W2.51,W2,52, W2,53, W2.54, W2.55, W2.56, W2.57, W2.58, W2.59, W2.60, W2.61,W2.62, W2.63, W2.64, W2.65, W2.66, W2.67, W2.68, W269, W270, W2.71,W2.72, W2.73, W2.74, W2.75, W2.76, W2.77, W2.78, W279, W2.80, W2.81,W2.82, W2.83, W2,84, W2.85, W2.86, W2.87, W2,88, W2,89, W2.90, andW2.91, or pharmaceutically acceptable salts thereof.

In certain embodiments, the ADC, or a pharmaceutically acceptable saltthereof, comprises a drug linked to an antibody by way of a linker,wherein the drug is a Bcl-xL inhibitor selected from the groupconsisting of W2.01, W2.02, W2.03, W2,04, W205, W2.06, W2.07, W2.08,W2,09, W2.10, W2.11, W2,12, W2.13, W2.14, W2.15, W2,16, W2.17, W2.18,W2.19, W2.20, W2,21, W2.22, W2.23, W224, W2.25, W2.26, W2.27, W2.28,W2.29, W2.30, W2.31, W2.32, W233, W2.34, W2.35, W2.36, W2.37, W2,38,W2.39, W2.40, W2.41, W2,42, W2,43, W2.44, W2.45, W2.46, W2.47, W2.48,W2.49, W2.50, W2.51, W2.52, W2.53, W2.54, W2.55, W2.56, W2.57, W2.58,W2.59, W260, W2.61, W2.62, W2.63, W2.64, W2.65, W2.66, W2.67, W2.68,W269, W2.70, W2.71, W2.72, W2.73, W2.74, W2.75, W2.76, W2.77, W2.78,W2.79, W2.80, W2.81, W2.82, W2.83, W2.84, W2.85, W286, W2.87, W2.88,W2.89, W290, and W2.91.

In certain embodiments, the ADC, or a pharmaceutically acceptable saltthereof, the Bcl-xL inhibitor is selected from the group consisting ofthe following compounds modified in that the hydrogen corresponding tothe # position of structural formula (IIa), (IIb), (IIc), or (IId) isnot present forming a monoradical:

-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(H)-yl]-3-[1-({3-[2-({2-[2-(carboxymethoxy)ethoxy]ethyl}amino)ethoxy]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(H)-yl]-3-{-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   2-{[(2-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]amino}ethyl)sulfonyl]amino}-2-deoxy-D-glucopyranose;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(4-{[(3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl]methyl}benzyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(3-sulfopropyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[(2,3-dihydroxypropyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   2-({[4-({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]amino}methyl)phenyl]sulfonyl}amino)-2-deoxy-beta-D-glucopyranose;-   8-(1,3-benzothiazol-2-ylcarbamoyl)-2-{6-carboxy-5-[1-({3-[2-({2-[1-(beta-D-glucopyranuronosyl)-1H-1,2,3-triazol-4-yl]ethyl}amino)ethoxy]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridin-2-yl}-1,2,3,4-tetrahydroisoquinoline;-   3-[1-({3-[2-(2-{[4-(beta-D-allopyranosyloxy)benzyl]amino}ethoxy)ethoxy]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3,5-dimethyl-7-(2-{2-[(2-sulfoethyl)amino]ethoxy}ethoxy)tricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-phosphonoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[methyl(3-sulfo-L-alanyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(3-phosphonopropyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(3-sulfo-L-alanyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3,5-dimethyl-7-(2-{2-[(3-phosphonopropyl)amino]ethoxy}ethoxy)tricyclo[3.3.1.1^(1,3)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylic    acid;-   3-{1-[(3-{2-[L-alpha-aspartyl(methyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylic    acid;-   6-{4-[({2-[2-(2-aminoethoxy)ethoxy]ethyl}[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]amino)methyl]benzyl}-2,6-anhydro-L-gulonic    acid;-   4-({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]amino}methyl)phenyl    hexopyranosiduronic acid;-   6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-phosphonoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]-3-{1-[(3,5-dimethyl-7-{2-[methyl(3-sulfo-L-alanyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-([1,3]thiazolo[5,4-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylic    acid;-   3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-([1,3]thiazolo[4,5-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylic    acid;-   6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[(2-carboxyethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(3-phosphonopropyl)(piperidin-4-yl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   3-{1-[(3-{2-[D-alpha-aspartyl(methyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3-(2-{[1-(carboxymethyl)piperidin-4-yl]amino}ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylic    acid;-   N-[(5S)-5-amino-6-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](methyl)amino}-6-oxohexyl]-N,N-dimethylmethanaminium;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[piperidin-4-yl(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methy]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-5-(3-phosphonopropoxy)-3,4-dihydroisoquinolin-2(1H)-yl]-3-[1-({3,5-dimethyl-7-[2-(methylamino)ethoxy]tricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3-(2-{[N-(2-carboxyethyl)-L-alpha-aspartyl]amino}ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylic    acid;-   3-{1-[(3-{2-[(2-aminoethyl)(2-sulfoethyl)amino]ethoxy}-57-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylic    acid;-   6-[5-(2-aminoethoxy)-8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-[1-({3,5-dimethyl-7-[2-(methylamino)ethoxy]tricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-3-{1-[(3,5-dimethyl-7-{2-[(3-sulfopropyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[(2-carboxyethyl)(piperidin-4-yl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(3-sulfo-L-alanyl)(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[2-[(2-carboxyethyl)amino]ethyl)(2-sulfoethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   3-{1-[(3,5-dimethyl-7-{2-[(3-phosphonopropyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-([1,3]thiazolo[4,5-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylic    acid;-   3-{1-[(3,5-dimethyl-7-{2-[(3-phosphonopropyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-([1,3]thiazolo[5,4-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-5-(carboxymethoxy)-3,4-dihydroisoquinolin-2(1H)-yl]-3-[1-({3,5-dimethyl-7-[2-(methylamino)ethoxy]tricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[(3-carboxypropyl)(piperidin-4-yl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-3-{I-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   3-{1-[(3-{2-[L-alpha-aspartyl(2-sulfoethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[(1,3-dihydroxypropan-2-yl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[5-(2-aminoethoxy)-8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[methyl(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-5-{2-[(2-sulfoethyl)amino]ethoxy}-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[methyl(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl){2-[(2-sulfoethyl)amino]ethyl}amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-5-{2-[(2-carboxyethyl)amino]ethoxy}-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[methyl(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   3-(1-[(3,5-dimethyl-7-{2-[(3-phosphonopropyl)(piperidin-4-yl)amino]ethoxy)tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-([1,3]thiazolo[4,5-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylic    acid;-   6-[4-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydro-2H-1,4-benzoxazin-6-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-5-(3-sulfopropoxy)-3,4-dihydroisoquinolin-2(1H)-yl]-3-[1-({3,5-dimethyl-7-[2-(methylamino)ethoxy]tricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylic    acid;-   3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[1-([1,3]thiazolo[4,5-b]pyridin-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]pyridine-2-carboxylic    acid;-   3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-([1,3]thiazolo[4,5-b]pyridin-2-ylcarbamoyl)naphthalen-2-yl]pyridine-2-carboxylic    acid;-   (1ζ)-1-({2-[5-(1-{[3-(2-aminoethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)-6-carboxypyridin-2-yl]-8-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroisoquinolin-5-yl})methyl)-1,5-anhydro-D-glucitol;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[(3-carboxypropyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-3-{1-[(3,5-dimethyl-7-{2-[(3-phosphonopropyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3-(2-{[4-(beta-D-glucopyranosyloxy)benzyl]amino}ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylic    acid;-   3-(1-{[3-(2-{[4-(beta-D-allopyranosyloxy)benzyl]amino}ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylic    acid;-   3-{1-[(3-{2-[azetidin-3-yl(2-sulfoethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylic    acid;-   3-{1-[(3-{2-[(3-aminopropyl)(2-sulfoethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylic    acid;-   6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]-3-{1-[(3-{2-[(2-carboxyethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[(N⁶,N⁶-dimethyl-L-lysyl)(methyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   3-{1-[(3-{2-[(3-aminopropyl)(methyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]pyridine-2-carboxylic    acid;-   3-{1-[(3-{2-[azetidin-3-yl(methyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]pyridine-2-carboxylic    acid;-   N⁶-(37-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-yl)-L-lysyl-N-[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]-L-alaninamide;-   methyl    6-[4-(3-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]amino}propyl)-1H-1,2,3-triazol-1-yl]-6-deoxy-beta-L-glucopyranoside;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-3-{1-[(3-{2-[(2-carboxyethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[5-(1,3-benzothiazol-2-ylcarbamoyl)quinolin-3-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[4-(1,3-benzothiazol-2-ylcarbamoyl)quinolin-6-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[5-(1,3-benzothiazol-2-ylcarbamoyl)quinolin-3-yl]-3-{1-[(3-{2-[(2-carboxyethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   8-(1,3-benzothiazol-2-ylcarbamoyl)-2-{6-carboxy-5-[1-({3-[2-({3-[1-(beta-D-glucopyranuronosyl)-1H-1.2.3-triazol-4-yl]propyl}amino)ethoxy]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridin-2-yl}-1,2,3,4-tetrahydroisoquinoline;-   6-[7-(1,3-benzothiazol-2-ylcarbamoyl)-1H-indol-2-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-6-[3-(methylamino)propyl]-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   5-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]amino}-5-deoxy-D-arabinitol;-   1-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]amino}-1,2-dideoxy-D-arabino-hexitol;-   6-[4-(1,3-benzothiazol-2-ylcarbamoyl)isoquinolin-6-yl]-3-{-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3-(2-{[3-hydroxy-2-(hydroxymethyl)propyl]amino}ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylic    acid;-   1-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]amino}-1,2-dideoxy-D-erythro-pentitol;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3,5-dimethyl-7-(2-{[(2S,3S)-2,3,4-trihydroxybutyl]amino}ethoxy)tricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3-(2-{[(2S,3S,4R,5R,6R)-2,3,4,5,6,7-hexahydroxyheptyl]amino}ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[({3-[(1,3-dihydroxypropan-2-yl)amino]propyl}sulfonyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[(3-{[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino}-3-oxopropyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3-(2-{[(3S)-3,4-dihydroxybutyl]amino}ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylic    acid;-   4-({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]amino}methyl)phenyl    beta-D-glucopyranosiduronic acid:-   3-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]amino}propyl    beta-D-glucopyranosiduronic acid;-   6-[4-(1,3-benzothiazol-2-ylcarbamoyl)-2-oxidoisoquinolin-6-yl]-3-[1-({3,5-dimethyl-7-[2-(methylamino)ethoxy]tricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylic    acid;-   6-{8-[(1,3-benzothiazol-2-yl)carbamoyl]-3,4-dihydroisoquinolin-2(1H)-yl}-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]acetamido}tricyclo[3.3.1.1^(3,7)]decan-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3,5-dimethyl-7-({2-[(2-sulfoethyl)amino]ethyl}sulfanyl)tricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylic    acid; and-   6-{8-[(1,3-benzothiazol-2-yl)carbamoyl]-3,4-dihydroisoquinolin-2(1H)-yl}-3-{1-[(3,5-dimethyl-7-{3-[(2-sulfoethyl)amino]propyl}tricyclo[3.3.1.1^(3,7)]decan-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;

and a pharmaceutically acceptable salt thereof.

The Bcl-xL inhibitors bind to and inhibit anti-apoptotic Bcl-xLproteins, inducing apoptosis. The ability of specific Bcl-xL inhibitorsaccording to structural formulae (IIa)-(IId) to bind to and inhibitBcl-xL activity may be confirmed in standard binding and activityassays, including, for example, the TR-FRET Bcl-xL binding assaysdescribed in Tao et al., 2014, ACS Med. Chem. Lett., 5:1088-1093, Aspecific TR-FRET Bcl-xL binding assay that can be used to confirm Bcl-xLbinding is provided in Example 4, below. Typically, Bcl-xL inhibitorsuseful as inhibitors per se and in the ADCs described herein willexhibit a K_(i) in the binding assay of Example 5 of less than about 1nM, but may exhibit a significantly lower K_(i), for example a K_(i) ofless than about 1, 0.1, or even 0.01 nM.

Bcl-xL inhibitory activity may also be confirmed in standard cell-basedcytotoxicity assays, such as the FL5.12 cellular and Molt-4 cytotoxicityassays described in Tao et al., 2014, ACS Med. Chem. Lett., 5:1088-1093.A specific Molt-4 cellular cytotoxicity assay that may be used toconfirm Bcl-xL inhibitory activity of specific Bcl-xL inhibitors thatare able to permeate cell membranes is provided in Examples 5 and 6,below. Typically, such cell-permeable Bcl-xL inhibitors will exhibit anEC₅₀ of less than about 500 nM in the Molt-4 cytotoxicity assay ofExamples 5 and 6, but may exhibit a significantly lower EC₅₀, forexample an EC₅₀ of less than about 250, 100, 50, 20, 10 or even 5 nM.

Owing to the presence of solubilizing groups, many of the Bcl-xLinhibitors described herein are expected to exhibit low or very low cellpermeability, and therefore will not yield significant activity incertain cellular assays due to the inability of the compound to traversethe cell membrane, including the Molt-4 cellular toxicity assay ofExamples 5 and 6. Bcl-xL inhibitory activity of compounds that do notfreely traverse cell membranes may be confirmed in cellular assays withpermeabilized cells. The process of mitochondrial outer-membranepermeabilization (MOMP) is controlled by the Bcl-2 family proteins.Specifically, MOMP is promoted by the pro-apoptotic Bcl-2 familyproteins Bax and Bak which, upon activation oligomerize on the outermitochondrial membrane and form pores, leading to release of cytochromec (cyt c). The release of cyt c triggers formulation of the apoptosomewhich, in turn, results in caspase activation and other events thatcommit the cell to undergo programmed cell death (see, Goldstein et al.,2005, Cell Death and Differentiation 12:453-462). The oligomerizationaction of Bax and Bak is antagonized by the anti-apoptotic Bcl-2 familymembers, including Bcl-2 and Bcl-xl-Bcl-xL inhibitors, in cells thatdepend upon Bcl-xL for survival, can cause activation of Bax and/or Bak,MOMP, release of cyt c and downstream events leading to apoptosis. Theprocess of cyt c release can be measured via western blot of bothmitochondrial and cytosolic fractions of cells and used as a proxymeasurement of apoptosis in cells.

As a means of detecting Bcl-xin inhibitory activity and consequentrelease of cyt c for Bcl-xL inhibitors with low cell permeability, thecells can be treated with an agent that causes selective pore formationin the plasma, but not mitochondrial, membrane. Specifically, thecholesterol/phospholipid ratio is much higher in the plasma membranethan the mitochondrial membrane. As a result, short incubation with lowconcentrations of the cholesterol-directed detergent digitoninselectively permeabilizes the plasma membrane without significantlyaffecting the mitochondrial membrane. This agent forms insolublecomplexes with cholesterol leading to the segregation of cholesterolfrom its normal phospholipid binding sites. This action, in turn, leadsto the formation of holes about 40-50 Å wide in the lipid bilayer. Oncethe plasma membrane is permeabilized, cytosolic components able to passover digitonin-formed holes can be washed out, including the cytochromeC that was released from mitochondria to cytosol in the apoptotic cells(Campos, 2006, Cytometry A 69(6):515-523).

Typically, Bcl-xL inhibitors will yield an EC₅₀ of less than about 10 nMin the Molt-4 cell permeabilized cyt c assay of Examples 5 and 6,although the compounds may exhibit significantly lower EC₅₀s, forexample, less than about 5, 1, or even 0.5 nM. As demonstrated inExample 6, Bcl-xL inhibitors having low or very low cell permeabilitythat do not exhibit activity in the standard Molt-4 cellular toxicityassay with non-permeablized cells exhibit potent functional activity, asmeasured by release of cyt c, in cellular cytotoxicity assays withpermeabilized cells. In addition to cytochrome c release, mitochondriaundergoing apoptosis frequently lose their transmembrane mitochondrialmembrane potential (Bouchier-Hayes et al., 2008, Methods 44(3):222-228). JC-1 is a cationic carbocyanine dye that accumulates inmitochondria and fluoresces red when mitochondria are healthy and islost when the mitochondrial membrane is compromised (percentagedepolarization; Smiley et al., 1991, Proc. Natl. Acad Sci. USA, 88:3671-3675; Reers et al., 1991: Biochemistry, 30: 4480-4486). This lossin signal can be detected in permeabilized cells using a fluorimeter(excitation 545 nm and emission of 590 nm) and is therefore fullyquantitative, enhancing both reproducibility and throughput. Typically,Bcl-xL inhibitors will yield an EC₅₀ of less than about 10 nM in theMolt-4 cell permeabilized JC-1 assay of Examples 5 and 6, although thecompounds may exhibit significantly lower EC₅₀s, for example, less thanabout 5, 1, 0,5 or even 0.05 nM, As demonstrated in Example 6, Bcl-xLinhibitors having low or very low cell permeability that do not exhibitactivity in the standard Molt-4 cellular toxicity assay withnon-permeablized cells exhibit potent functional activity, as measuredby their loss of transmembrane mitochondrial membrane potential in theJC-1 assay, in cellular cytotoxicity assays with permeabilized cells.Low permeability Bcl-xL inhibitors also exhibit potent activity whenadministered to cells in the form of ADCs (see, e.g., Example 8).

Although many of the Bcl-xL inhibitors of structural formulae(IIa)-(IId) selectively or specifically inhibit Bcl-xL over otheranti-apoptotic Bcl-2 family proteins, selective and/or specificinhibition of Bcl-xL is not necessary. The Bcl-xL inhibitors and ADCscomprising the compounds may also, in addition to inhibiting Bcl-xL,inhibit one or more other anti-apoptotic Bcl-2 family proteins, such as,for example, Bcl-2. In some embodiments, the Bcl-xL inhibitors and/orADCs are selective and/or specific for Bcl-xL. By specific or selectiveis meant that the particular Bcl-xL inhibitor and/or ADC binds orinhibits Bcl-xL to a greater extent than Bcl-2 under equivalent assayconditions. In specific embodiments, the Bcl-xL inhibitors and/or ADCsexhibit in the range of about 10-fold, 100-fold, or even greaterspecificity or selectivity for Bcl-xL than Bcl-2 in binding assays.

III.A.2.Bcl-xL Linkers

In the ADCs described herein, the Bcl-xL inhibitors are linked to theantibody by way of linkers. The linker linking a Bcl-xL inhibitor to theantibody of an ADC may be short, long, hydrophobic, hydrophilic,flexible or rigid, or may be composed of segments that eachindependently has one or more of the above-mentioned properties suchthat the linker may include segments having different properties. Thelinkers may be polyvalent such that they covalently link more than oneBcl-xL inhibitor to a single site on the antibody, or monovalent suchthat covalently they link a single Bcl-xL, inhibitor to a single site onthe antibody.

As will be appreciated by skilled artisans, the linkers link the Bcl-xLinhibitors to the antibody by forming a covalent linkage to the Bcl-xLinhibitor at one location and a covalent linkage to antibody at another.The covalent linkages are formed by reaction between functional groupson the linker and functional groups on the inhibitors and antibody. Asused herein, the expression “linker” is intended to include (i)unconjugated forms of the linker that include a functional group capableof covalently linking the linker to a Bcl-xL inhibitor and a functionalgroup capable of covalently linking the linker to an antibody; (ii)partially conjugated forms of the linker that include a functional groupcapable of covalently linking the linker to an antibody and that iscovalently linked to a Bcl-xL inhibitor, or vice versa; and (iii) fullyconjugated forms of the linker that is covalently linked to both aBcl-xL, inhibitor and an antibody. In some specific embodiments ofintermediate synthons and ADCs described herein, moieties comprising thefunctional groups on the linker and covalent linkages formed between thelinker and antibody are specifically illustrated as R^(x) and LK,respectively.

The linkers are preferably, but need not be, chemically stable toconditions outside the cell, and may be designed to cleave, immolateand/or otherwise specifically degrade inside the cell. Alternatively,linkers that are not designed to specifically cleave or degrade insidethe cell may be used. A wide variety of linkers useful for linking drugsto antibodies in the context of ADCs are known in the art. Any of theselinkers, as well as other linkers, may be used to link the Bcl-xLinhibitors to the antibody of the ADCs described herein.

Exemplary polyvalent linkers that may be used to link many Bcl-xLinhibitors to an antibody are described, for example, in U.S. Pat. No.8,399,512; U.S. Published Application No. 2010/0152725; U.S. Pat. Nos.8,524,214; 8,349,308; U.S. Published Application No. 2013/189218; U.S.Published Application No. 2014/017265; WO 2014/093379; WO 2014/093394;WO 2014/093640, the contents of which are incorporated herein byreference in their entireties. For example, the Fleximer® linkertechnology developed by Mersana et al. has the potential to enablehigh-DAR ADCs with good physicochemical properties. As shown below, theFleximer® linker technology is based on incorporating drug moleculesinto a solubilizing poly-acetal backbone via a sequence of ester bonds.The methodology renders highly-loaded ADCs (DAR up to 20) whilstmaintaining good physicochemical properties. This methodology could beutilized with Bcl-xL inhibitors as shown in the Scheme below.

To utilize the Fleximer® linker technology depicted in the scheme above,an aliphatic alcohol can be present or introduced into the Bcl-xLinhibitor. The alcohol moiety is then conjugated to an alanine moiety,which is then synthetically incorporated into the Fleximer® linker.Liposomal processing of the ADC in vitro releases the parentalcohol-containing drug.

Additional examples of dendritic type linkers can be found in US2006/116422; US 2005/271615; de Groot et al, (2003) Angew. Chem. Int. Ed42:4490-4494; Amir et al, (2003) Angew. Chem. Mt. Ed, 42:4494-4499;Shamis et al., (2004) J. Am. Chem. Soc. 126:1726-1731; Sun et al.,(2002) Bioorganic & Medicinal Chemistry letters 12:2213-2215; Sun etal., (2003) Bioorganic Medicinal Chemistry 11:1761-1768; King et al,(2002) Tetrahedron Letters 43:1987-1990.

Exemplary monovalent linkers that may be used are described, forexample, in Nolting, 2013, Antibody-Drug Conjugates, Methods inMolecular Biology 1045:71-100; Kitson et al, 2013, CROs/CMOs—ChemicaOggi—Chemistry Today 31(4): 30-36; Ducry et al., 2010, BioccmjugateChem. 21:5-13; Zhao et al., 2011, J. Med. Chem. 54:3606-3623; U.S. Pat.Nos. 7,223,837; 8,568,728; 8,535,678; and 02004010957, the content ofeach of which is incorporated herein by reference in their entireties.

By way of example and not limitation, some cleavable and noncleavablelinkers that may be included in the ADCs described herein are describedbelow.

Cleavable Linkers

In certain embodiments, the linker selected is cleavable in vitro and invivo. Cleavable linkers may include chemically or enzymatically unstableor degradable linkages. Cleavable linkers generally rely on processesinside the cell to liberate the drug, such as reduction in thecytoplasm, exposure to acidic conditions in the lysosome, or cleavage byspecific proteases or other enzymes within the cell. Cleavable linkersgenerally incorporate one or more chemical bonds that are eitherchemically or enzymatically cleavable while the remainder of the linkeris noncleavable.

In certain embodiments, a linker comprises a chemically labile groupsuch as hydrazone and/or disulfide groups. Linkers comprising chemicallylabile groups exploit differential properties between the plasma andsome cytoplasmic compartments. The intracellular conditions tofacilitate drug release for hydrazone containing linkers are the acidicenvironment of endosomes and lysosomes, while the disulfide containinglinkers are reduced in the cytosol, which contains high thiolconcentrations, e.g., glutathione In certain embodiments, the plasmastability of a linker comprising a chemically labile group may beincreased by introducing steric hindrance using substituents near thechemically labile group.

Acid-labile groups, such as hydrazone, remain intact during systemiccirculation in the blood's neutral pH environment (pH 7.3-7.5) andundergo hydrolysis and release the drug once the ADC is internalizedinto mildly acidic endosomal (pH 5.0-6,5) and lysosomal (pH 4.5-5.0)compartments of the cell. This pH dependent release mechanism has beenassociated with nonspecific release of the drug. To increase thestability of the hydrazone group of the linker, the linker may be variedby chemical modification, e.g, substitution, allowing tuning to achievemore efficient release in the lysosome with a minimized loss incirculation.

Hydrazone-containing linkers may contain additional cleavage sites, suchas additional acid-labile cleavage sites and/or enzymatically labilecleavage sites. ADCs including exemplary hydrazone-containing linkersinclude the following structures:

wherein D and Ab represent the drug and Ab, respectively, and nrepresents the number of drug-linkers linked to the antibody. In certainlinkers such as linker (Ig), the linker comprises two cleavable groups—adisulfide and a hydrazone moiety. For such linkers, effective release ofthe unmodified free drug requires acidic pH or disulfide reduction andacidic pH. Linkers such as (Ih) and (II) have been shown to be effectivewith a single hydrazone cleavage site.

Other acid-labile groups that may be included in linkers includecis-aconityl-containing linkers. cis-Aconityl chemistry uses acarboxylic acid juxtaposed to an amide bond to accelerate amidehydrolysis under acidic conditions.

Cleavable linkers may also include a disulfide group. Disulfides arethermodynamically stable at physiological pH and are designed to releasethe drug upon internalization inside cells, wherein the cytosol providesa significantly more reducing environment compared to the extracellularenvironment. Scission of disulfide bonds generally requires the presenceof a cytoplasmic thiol cofactor, such as (reduced) glutathione (GSH),such that disulfide-containing linkers are reasonable stable incirculation, selectively releasing the drug in the cytosol. Theintracellular enzyme protein disulfide isomerase, or similar enzymescapable of cleaving disulfide bonds, may also contribute to thepreferential cleavage of disulfide bonds inside cells. GSH is reportedto be present in cells in the concentration range of 0.5-10 mM comparedwith a significantly lower concentration of GSH or cysteine, the mostabundant low-molecular weight thiol, in circulation at approximately 5μM. Tumor cells, where irregular blood flow leads to a hypoxic state,result in enhanced activity of reductive enzymes and therefore evenhigher glutathione concentrations. In certain embodiments, the in vivastability of a disulfide-containing linker may be enhanced by chemicalmodification of the linker, e.g., use of steric hindrance adjacent tothe disulfide bond.

ADCs including exemplary disulfide-containing linkers include thefollowing structures:

wherein D and Ab represent the drug and antibody, respectively, nrepresents the number of drug-linkers linked to the antibody and R isindependently selected at each occurrence from hydrogen or alkyl, forexample. In certain embodiments, increasing steric hindrance adjacent tothe disulfide bond increases the stability of the linker. Structuressuch as (Ij) and (Il) show increased, in viva stability when one or moreR groups is selected from a lower alkyl such as methyl.

Another type of linker that may be used is a linker that is specificallycleaved by an enzyme. Such linkers are typically peptide-based orinclude peptidic regions that act as substrates for enzymes. Peptidebased linkers tend to be more stable in plasma and extracellular milieuthan chemically labile linkers. Peptide bonds generally have good serumstability, as lysosomal proteolytic enzymes have very low activity inblood clue to endogenous inhibitors and the unfavorably high pH value ofblood compared to lysosomes. Release of a drug from an antibody occursspecifically due to the action of lysosomal proteases, e.g., cathepsinand plasmin. These proteases may be present at elevated levels incertain tumor tissues. In certain embodiments, the linker is cleavableby a lysosomal enzyme. In certain embodiments, the linker is cleavableby a lysosomal enzyme, and the lysosomal enzyme is Cathepsin B. Incertain embodiments, the linker is cleavable by a lysosomal enzyme, andthe lysosomal enzyme is β-glucuronidase or β-galactosidase. In certainembodiments, the linker is cleavable by a lysosomal enzyme, and thelysosomal enzyme is β-glucuronidase. In certain embodiments, the linkeris cleavable by a lysosomal enzyme, and the lysosomal enzyme isβ-galactosidase.

Those skilled in the art recognize the importance of cleavable linkersthat are stable to plasma, yet are readily cleaved by a lysosomalenzyme. Disclosed herein, in certain embodiments, are linkers, cleavableby the lysosomal enzymes β-glucuronidase or β-galactosidase, that showimproved plasma stability and reduced non-specific release of smallmolecule drug.

In exemplary embodiments, the cleavable peptide is selected fromtetrapeptides such as Gly-Phe-Len-Gly, Ala-Leu-Ala-Leu or dipeptidessuch as Val-Cit, Val-Ala, and Phe-Lys. In certain embodiments,dipeptides are preferred over longer polypeptides due to hydrophobicityof the longer peptides.

A variety of dipeptide-based cleavable linkers useful for linking drugssuch as doxorubicin, mitomycin, camptothecin, tallysomycin andauristatin/auristatin family members to antibodies have been described(see, Dubowchik et al., 1998, J. Org. Chem. 67:1866-1872; Dubowchik etat, 1998, Bioorg. Med. Chem. Led 8:3341-3346; Walker et al., 2002,Bioorg. Med. Chern. Lett. 12:217-219; Walker et at, 2004, Bioorg. Med.Chem. Lett 14:4323-4327, and Francisco et al., 2003, Blood102:1458-1465, the contents of each of which are incorporated herein byreference). All of these dipeptide linkers, or modified versions ofthese dipeptide linkers, may be used in the ADCs described herein, Otherdipeptide linkers that may be used include those found in ADCs such asSeattle Genetics' Brentuximab Vendotin SGN-35 (Adcetris™), SeattleGenetics SGN-75 (anti-CD-70, MC-monomethyl auristatin F(MMAF), CelldexTherapeutics glembatutnumab (CDX-011) (anti-NMB, Val-Cit-monomethylauristatin E(MMAE), and Cytogen PSMA-ADC (PSMA-ADC-1301) (anti-PSMA,Val-Cit-MMAE).

Enzymatically cleavable linkers may include a self-immolative spacer tospatially separate the drug from the site of enzymatic cleavage. Thedirect attachment of a drug to a peptide linker can result inproteolytic release of an amino acid adduct of the drug therebyimpairing its activity. The use of a self-immolative spacer allows forthe elimination of the fully active, chemically unmodified drug uponamide bond hydrolysis.

One self-immolative spacer is the bifunctional para-aminobenzyl alcoholgroup, which is linked to the peptide through the amino group, formingan amide bond, while amine containing drugs may be attached throughcarbamate functionalities to the benzylic hydroxyl group of the linker(to give a p-amidobenzylcarbamate, PABC). The resulting prodrugs areactivated upon protease-mediated cleavage, leading to a 1,6-eliminationreaction releasing the unmodified drug, carbon dioxide, and remnants ofthe linker group. The following scheme depicts the fragmentation ofp-amidobenzyl carbamate and release of the drug:

wherein X-D represents the unmodified drug. Heterocyclic variants ofthis self-immolative group have also been described. See U.S. Pat. No.7,989,434.

In certain embodiments, the enzymatically cleavable linker is aß-glucuronic acid-based linker. Facile release of the drug may berealized through cleavage of the ß-glucuronide glycosidic bond by thelysosomal enzyme ß-glucuronidase. This enzyme is present abundantlywithin lysosomes and is overexpressed in some tumor types, while theenzyme activity outside cells is low, ß-Glucuronic acid-based linkersmay be used to circumvent the tendency of an ADC to undergo aggregationdue to the hydrophilic nature of ß-glucuronides. In certain embodiments,ß-glucuronic acid-based linkers are preferred as linkers for ADCs linkedto hydrophobic drugs. The following scheme depicts the release of thedrug from and ADC containing a ß-glucuronic acid-based linker:

A variety of cleavable 6-glucuronic acid-based linkers useful forlinking drugs such as auristatins, camptothecin and doxorubicinanalogues, CBI minor-groove binders, and psymberin to antibodies havebeen described (see, Jeffrey et al., 2006, Bioconjug. Chem. 17:831-840;Jeffrey et al., Bioorg. Med. Chem. Lett. 17:2278-2280; and Jiang et al.,2005, J. Am. Chem. Soc. 127:11254-11255, the contents of each of whichare incorporated herein by reference). All of these ß-glucuronicacid-based linkers may be used in the ADCs described herein. In certainembodiments, the enzymatically cleavable linker is a ß-galactoside-basedlinker. ß-Galactoside is present abundantly within lysosomes, while theenzyme activity outside cells is low. Additionally, Bcl-xL inhibitorscontaining a phenol group can be covalently bonded to a linker throughthe phenolic oxygen. One such linker, described in U.S. Published App.No. 2009/0318668, relies on a methodology in which a diamino-ethane“SpaceLink” is used in conjunction with traditional “PABO”-basedself-immolative groups to deliver phenols. The cleavage of the linker isdepicted schematically below using a Bcl-xL inhibitor of the disclosure.

Cleavable linkers may include noncleavable portions or segments, and/orcleavable segments or portions may be included in an otherwisenon-cleavable linker to render it cleavable. By way of example only,polyethylene glycol (PEG) and related polymers may include cleavablegroups in the polymer backbone. For example, a polyethylene glycol orpolymer linker may include one or more cleavable groups such as adisulfide, a hydrazone or a dipeptide.

Other degradable linkages that may be included in linkers include esterlinkages formed by the reaction of PEG carboxylic acids or activated PEGcarboxylic acids with alcohol groups on a biologically active agent,wherein such ester groups generally hydrolyze under physiologicalconditions to release the biologically active agent. Hydrolyticallydegradable linkages include, but are not limited to, carbonate linkages;mime linkages resulting from reaction of an amine and an aldehyde;phosphate ester linkages formed by reacting an alcohol with a phosphategroup; acetal linkages that are the reaction product of an aldehyde andan alcohol; orthoester linkages that are the reaction product of aformate and an alcohol; and oligonucleotide linkages formed by aphosphoramidite group, including but not limited to, at the end of apolymer, and a 5 hydroxyl group of an oligonucleotide.

In certain embodiments, the linker comprises an enzymatically cleavablepeptide moiety, for example, a linker comprising structural formula(IVa), (IVb), (IVc) or (IVd):

or a pharmaceutically acceptable salt thereof, wherein:

-   -   peptide represents a peptide (illustrated N→C, wherein peptide        includes the amino and carboxy “termini”) cleavable by a        lysosomal enzyme;    -   T represents a polymer comprising one or more ethylene glycol        units or an alkylene chain, or combinations thereof.    -   R^(a) is selected from hydrogen, C₁₋₆ alkyl, SO₃H and CH₂SO₃H;    -   R^(y) is hydrogen or C₁₋₄ alkyl-(O)_(r)—(C₁₋₄ alkylene)_(s)-G¹        or C₁₋₄ alkyl-(N)—[(C₁₋₄ alkylene)-G¹]₂;    -   R^(z) is C₁₋₄ alkyl-(O)_(r)—(C₁₋₄ alkylene)_(s)-G²;        -   G¹ is SO₃H, CO₂H, PEG 4-32, or sugar moiety;        -   G² is SO₃H, CO₂H, or PEG 4-32 moiety;        -   r is 0 or 1;        -   s is 0 or 1;        -   p is an integer ranging from 0 to 5;        -   q is 0 or 1;        -   x is 0 or 1;        -   y is 0 or 1;        -   represents the point of attachment of the linker to the            Bcl-xL inhibitor; and        -   * represents the point of attachment to the remainder of the            linker.

In certain embodiments, the linker comprises an enzymatically cleavablepeptide moiety, for example, a linker comprising structural formula(IVa), (IVb), (IVc), or (IVd), or a pharmaceutically acceptable saltthereof.

In certain embodiments, the peptide is selected from a tripeptide or adipeptide. In particular embodiments, the dipeptide is selected from:Val-Cit; Cit-Val; Ala-Ala; Ala-Cit; Cit-Ala; Asn-Cit; Cit-Asn; Cit-Cit;Val-Glu; Glu-Val; Ser-Cit; Cit-Ser; Lys-Cit; Cit-Lys; Asp-Cit; Cit-Asp;Ala-Val; Val-Ala; Phe-Lys; Lys-Phe; Val-Lys; Lys-Val; Ala-Lys; Lys-Ala;Phe-Cit; Cit-Phe; Leu-Cit; Cit-Leu; Ile-Cit; Cit-Ile; Phe-Arg; Arg-Phe;Cit-Trp; and Trp-Cit; or a pharmaceutically acceptable salt thereof.

Exemplary embodiments of linkers according to structural formula (IVa)that may be included in the ADCs described herein include the linkersillustrated below (as illustrated, the linkers include a group suitablefor covalently linking the linker to an antibody):

Exemplary embodiments of linkers according to structural formula (IVb),(IVc), or (IVd) that may be included in the ADCs described hereininclude the linkers illustrated below (as illustrated, the linkersinclude a group suitable for covalently linking the linker to anantibody):

In certain embodiments, the linker comprises an enzymatically cleavablesugar moiety, for example, a linker comprising structural formula (Va),(Vb), (Vc), (Vd), or (Ve):

or a pharmaceutically acceptable salt thereof; wherein:

-   -   q is 0 or 1;    -   r is 0 or 1;    -   X¹ is CH₂, O or NH;    -   represents the point of attachment of the linker to the drug;        and    -   represents the point of attachment to the remainder of the        linker.

Exemplary embodiments of linkers according to structural formula (Va)that may be included in the ADCs described herein include the linkersillustrated below as illustrated, the linkers include a group suitablefor covalently linking; the linker to an antibody):

Exemplary embodiments of linkers according to structural formula (Vb)that may be included in the ADCs described herein include the linkersillustrated below as illustrated, the linkers include a group suitablefor covalently linking the linker to an antibody):

Exemplary embodiments of linkers according to structural formula (Vc)that may be included in the ADCs described herein include the linkersillustrated below (as illustrated, the linkers include a group suitablefor covalently linking the linker to an antibody):

Exemplary embodiments of linkers according to structural formula (Vd)that may be included in the ADCs described herein include the linkersillustrated below (as illustrated, the linkers include a group suitablefor covalently linking the linker to an antibody):

Exemplary embodiments of linkers according to structural formula (Ve)that may be included in the ADCs described herein include the linkersillustrated below (as illustrated, the linkers include a group suitablefor covalently linking the linker to an antibody):

Non-Cleavable Linkers

Although cleavable linkers may provide certain advantages, the linkerscomprising the ADC described herein need not be cleavable. Fornoncleavable linkers, the drug release does not depend on thedifferential properties between the plasma and some cytoplasmiccompartments. The release of the drug is postulated to occur afterinternalization of the ADC via antigen-mediated endocytosis and deliveryto lysosomal compartment, where the antibody is degraded to the level ofamino acids through intracellular proteolytic degradation. This processreleases a drug derivative, which is formed by the drug, the linker, andthe amino acid residue to which the linker was covalently attached. Theamino-acid drug metabolites from conjugates with noncleavable linkersare more hydrophilic and generally less membrane permeable, which leadsto less bystander effects and less nonspecific toxicities compared toconjugates with a cleavable linker. In general, ADCs with noncleavablelinkers have greater stability in circulation than ADCs with cleavablelinkers. Non-cleavable linkers may be alkylene chains, or maybepolymeric in natures, such as, for example, based upon polyalkyleneglycol polymers, amide polymers, or may include segments of alkylenechains, polyalkylene glycols and/or amide polymers. In certainembodiments, the linker comprises a polyethylene glycol segment havingfrom 1 to 6 ethylene glycol units.

A variety of non-cleavable linkers used to link drugs to antibodies havebeen described. (See, Jeffrey et al., 2006, Bioconjug. Chem. 17;831-840; Jeffrey et al., 2007, Bioorg. Med. Chem. Lett. 17:2278-2280;and Jiang et al., 2005, J. Am Chem. Soc. 127:11254-11255, the contentsof which are incorporated herein by reference). All of these linkers maybe included in the ADCs described herein.

In certain embodiments, the linker is non-cleavable in vivo, for examplea linker according to structural formula (VIa), (VIb), (VIc) or (VId)(as illustrated, the linkers include a group suitable for covalentlylinking the linker to an antibody:

or a pharmaceutically acceptable salt thereof wherein:

R^(a) is selected from hydrogen, alkyl, sulfonate and methyl sulfonate;

R^(x) is a moiety including a functional group capable of covalentlylinking the linker to an antibody; and

represents the point of attachment of the linker to the Bcl-xLinhibitor.

Exemplary embodiments of linkers according to structural formula(VIa)-(VId) that may be included in the ADCs described herein includethe linkers illustrated below (as illustrated, the linkers include agroup suitable for covalently linking the linker to an antibody, and “

” represents the point of attachment to a Bcl-xL inhibitor):

Groups Used to Attach Linkers to Anti-B7-H3 Antibodies

Attachment groups can be electrophilic in nature and include: maleimidegroups, activated disulfides, active esters such as NHS esters and HOBtesters, haloformates, acid halides, alkyl and benzyl halides such ashaloacetamides. As discussed below, there are also emerging technologiesrelated to “self-stabilizing” maleimides and “bridging disulfides” thatcan be used in accordance with the disclosure.

Loss of the drug-linker from the ADC has been observed as a result of amaleimide exchange process with albumin, cysteine or glutathione (Alleyet al., 2008, Bioconjugate Chem. 19: 759-769). This is particularlyprevalent from highly solvent-accessible sites of conjugation whilesites that are partially accessible and have a positively chargedenvironment promote maleimide ring hydrolysis (Junutula et al., 2008,Nat. Biotechnol. 26: 925-932). A recognized solution is to hydrolyze thesuccinimide formed from conjugation as this is resistant todeconjugation from the antibody, thereby making the ADC stable in serum.It has been reported previously that the succinimide ring will undergohydrolysis under alkaline conditions (Kalia et al., 2007, Bioorg. Med.Chem. Lett. 17: 6286-6289). One example of a “self-stabilizing”maleimide group that hydrolyzes spontaneously under antibody conjugationconditions to give an ADC species with improved stability is depicted inthe schematic below. See U.S. Published Application No. 2013/0309256,International Application Publication No. WO 2013/173337, Tumey et al.,2014, Bioconjugate Chem. 25: 1871-1880, and Lyon et al., 2014, Nat.Biotechnol. 32: 1059-1062. Thus, the maleimide attachment group isreacted with a sulfhydryl of an antibody to give an intermediatesuccinimide ring. The hydrolyzed form of the attachment group isresistant to deconjugation in the presence of plasma proteins.

As shown above, the maleimide ring of a linker may react with anantibody Ab, forming a covalent attachment as either a succinimide(closed form) or succinamide (open form).

Polytherics has disclosed a method for bridging a pair of sulfhydrylgroups derived from reduction of a native hinge disulfide bond. See,Badescu et al., 2014, Bioconjugate Chem. 25:1124-1136. The reaction isdepicted in the schematic below. An advantage of this methodology is theability to synthesize homogenous DAR4 ADCs by full reduction of IgGs (togive 4 pairs of sulfhydryls) followed by reaction with 4 equivalents ofthe alkylating agent. ADCs containing “bridged disulfides” are alsoclaimed to have increased stability.

Similarly, as depicted below, a maleimide derivative that is capable ofbridging a pair of sulfhydryl groups has been developed. See U.S.Published Application No. 2013/0224228.

In certain embodiments the attachment moiety comprises the structuralformulae (VIIa), (VIIb), or (VIIc):

or a pharmaceutically acceptable salt thereof, wherein:

R^(q) is H or O—(CH₂CH₂O)₁₁—CH₃;

x is 0 or 1;

y is 0 or 1;

G³ is CH₂CH₂CH₂SO₃H or CH₂CH₂O—(CH₂CH₂O)₁₁—CH₃;

R^(w) is O—CH₂CH₂SO₃H or NH(CO)—CH₂CH₂O—(CH₂CH₂O)₁₂—CH₃; and

* represents the point of attachment to the remainder of the linker.

In certain embodiments, the linker comprises a segment according tostructural formulae (VIIIa), (YIIIb), or (VIIIc):

or a hydrolyzed derivative or a pharmaceutically acceptable saltthereof, wherein:

R^(q) is H or —O—(CH₂CH₂O)₁₁—CH₃.

x is 0 or 1;

y is 0 or 1;

G³ is —CH₂CH₂CH₂SO₃H or —CH₂CH₂O—(CH₂CH₂O)₁₁—CH₃.

R^(w) is —O—CH₂CH₂SO₃H or —NH(CO)—CH₂CH₂O—(CH₂CH₂O)₁₂—CH₃;

* represents the point of attachment to the remainder of the linker; and

represents the point of attachment of the linker to the antibody.

Exemplary embodiments of linkers according to structural formula (VIIa)and (VIIb) that may be included in the ADCs described herein include thelinkers illustrated below (as illustrated, the linkers include a groupsuitable for covalently linking the linker to an antibody):

Exemplary embodiments of linkers according to structural formula (Vile)that may be included in the ADCs described herein include the linkersillustrated below (as illustrated, the linkers include a group suitablefor covalently linking the linker to an antibody):

In certain embodiments, L is selected from the group consisting ofIVa.1-IVa.8, IVb.1, IVb.19, IVc.1-IVc.7, IVd.1-IVd.4, Va.1-Va.12,Vb.1-Vb.10, Vc.1-Vc.11, Vd.1-Vd.6, Ve.1-Ve.2, VIa.1, VIc.1-VIc.2,VID.1-VId.4. VIIa.1-VIIa.4, VIIb1-VIIb.8, VIIc.1-VIIc.6 in either theclosed or open form, and a pharmaceutically acceptable salt thereof.

In certain embodiments, L is selected from the group consisting ofIVb.2, IVc.5, IVc.6, IVc.7, IVd.4, Vb.9, VIIa.1, VIIa.3, VIIc.1, VIIc.4,and VIIc.5, wherein the maleimide of each linker has reacted with theantibody Ab, forming a covalent attachment as either a succinimide(closed form) or succinamide (open form),and a pharmaceuticallyacceptable salt thereof.

In certain embodiments, L is selected from the group consisting ofIVb.2, IVc.5, IVc.6, IVd.4, VIIa.1, VIIa.3, VIIc.1, VIIc.4, VIIc.5,wherein the maleimide of each linker has reacted with the antibody Ab,forming a covalent attachment as either a succinimide (closed Rum) orsuccinamide (open form), and a pharmaceutically acceptable salt thereof.

In certain embodiments, L is selected from the group consisting ofIVb.2, VIIa.3, IVc.6, and VIIc.1, wherein

is the attachment point to drug D and @ is the attachment point to theLK, wherein when the linker is in the open form as shown below, @ can beeither at the α-position β-position of the carboxylic acid next to it:

Bcl-xL Linker Selection Considerations

As is known by skilled artisans, the linker selected for a particularADC may be influenced by a variety of factors, including but not limitedto, the site of attachment to the antibody (e.g., lys, cys or otheramino acid residues), structural constraints of the drug pharmacophoreand the lipophilicity of the drug. The specific linker selected for anADC should seek to balance these different factors for the specificantibody/drug combination. For a review of the factors that areinfluenced by choice of linkers in ADCs, see Nolan, Chapter 5 “LinkerTechnology in Antibody-Drug Conjugates,” In: Antibody-Drug Conjugates:Methods in Molecular Biology, vol, 1045, pp. 71-100, Laurent Duery(Ed.), Springer Science & Business Medica, LLC, 2013.

For example, ADCs have been observed to effect killing of bystanderantigen-negative cells present in the vicinity of the antigen-positivetumor cells. The mechanism of bystander cell killing by ADCs hasindicated that metabolic products formed during intracellular processingof the ADCs may play a role. Neutral cytotoxic metabolites generated bymetabolism of the ADCs in antigen-positive cells appear to play a rolein bystander cell killing while charged metabolites may be preventedfrom diffusing across the membrane into the medium and therefore cannotaffect bystander killing. In certain embodiments, the linker is selectedto attenuate the bystander killing effect caused by cellular metabolitesof the ADC. In certain embodiments, the linker is selected to increasethe bystander killing effect.

The properties of the linker may also impact aggregation of the ADCunder conditions of use and/or storage. Typically, ADCs reported in theliterature contain no more than 3-4 drug molecules per antibody molecule(see, e.g., Chari, 2008, Acc Chem Res 41:98-107). Attempts to obtainhigher drug-to-antibody ratios (“DAR”) often failed, particularly ifboth the drug and the linker were hydrophobic, due to aggregation of theADC (see King et al., 2002, J Med Chem 45:4336-4343; Hollander et al.,2008, Bioconjugate Chem 19:358-361; Burke et al., 2009 Bioconjugate Chem20:1242-1250). In many instances, DARs higher than 3-4 could bebeneficial as a means of increasing potency. In instances where theBcl-xL inhibitor is hydrophobic in nature, it may be desirable to selectlinkers that are relatively hydrophilic as a means of reducing ADCaggregation, especially in instances where DARS greater than 3-4 aredesired. Thus, in certain embodiments, the linker incorporates chemicalmoieties that reduce aggregation of the ADCs during storage and/or use.A linker may incorporate polar or hydrophilic groups such as chargedgroups or groups that become charged under physiological pH to reducethe aggregation of the ADCs. For example, a linker may incorporatecharged groups such as salts or groups that deprotonate, carboxylates,or protonate, e.g., amines, at physiological pH.

Exemplary polyvalent linkers that have been reported to yield DARs ashigh as 20 that may be used to link numerous Bcl-xL inhibitors to anantibody are described in U.S. Pat. No. 8,399,512; U.S. PublishedApplication No. 2010/0152725; U.S. Pat. Nos. 8,524,214; 8,349,308; U.S.Published Application No, 2013/189218; U.S. Published Application No.2014/017265; WO 2014/093379; WO 2014/093394; WO 2014/093640, the contentof which are incorporated herein by reference in their entireties.

In particular embodiments, the aggregation of the ADCs during storage oruse is less than about 40% as determined by size-exclusionchromatography (SEC). In particular embodiments, the aggregation of theADCs during storage or use is less than 35%, such as less than about30%, such as less than about 25%, such as less than about 20%, such asless than about 15%, such as less than about 10%, such as less thanabout 5%, such as less than about 4%, or even less, as determined bysize-exclusion chromatography (SEC).

II.A.3. ADC Synthons

Antibody-Drug Conjugate synthons are synthetic intermediates used to foru ADCs. The synthons are generally compounds according to structuralformula (III):

D-L-R^(x)  (III)

or a pharmaceutically acceptable salt thereof, wherein D is a Bcl-xLinhibitor as previously described, L is a linker as previouslydescribed, and R^(x) is a reactive group suitable for linking thesynthon to an antibody.

In specific embodiments, the intermediate synthons are compoundsaccording to structural formulae (IIIa), (IIIb), (IIIc) and (IIId),below, or a pharmaceutically acceptable salt thereof, where the varioussubstituents Ar¹, A^(r2), Z¹, Z^(2a), Z^(2b), R′, R¹, R², R⁴, R^(11a),R^(11b), R¹² and R¹³ are as previously defined for structural formulae(IIa), (IIb), (IIc) and (IId), respectively, L is a linker as previouslydescribed and R^(x) is a functional group as described above:

To synthesize an ADC, an intermediate synthon according to structuralformula (III), or a salt thereof, is contacted with an antibody ofinterest under conditions in which functional group R^(x) reacts with a“complementary” functional group on the antibody, F^(x), to form acovalent linkage.

D-L-R^(x)+[F^(x)_(m)Ab→(I)[D-L-LK_(m)Ab  (III)

The identities of groups R^(x) and F^(x) will depend upon the chemistryused to link the synthon to the antibody. Generally, the chemistry usedshould not alter the integrity of the antibody, for example its abilityto bind its target. Preferably, the binding properties of the conjugatedantibody will closely resemble those of the unconjugated antibody. Avariety of chemistries and techniques for conjugating molecules tobiological molecules such as antibodies are known in the art and inparticular to antibodies, are well-known. See, e.g., Amon et al.,“Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,”in Monoclonal Antibodies And Cancer Therapy, Reisfeld et at Eds. Alan R.Liss. Inc., 1985; Hellstrom et al., “Antibodies For Drug Delivery.” inControlled Drug Delivery, Robinson et al., Eds., Marcel Dekker. Inc.,2nd Ed 1987; Thorpe. “Antibody Carriers Of Cytotoxic Agents In CancerTherapy A Review,” in: Monoclonal Antibodies '84: Biological AndClinical Applications. Pinchers et al., Eds. 1985; “Analysis, Results,and Future Prospective of the Therapeutic Use of Radiolabeled AntibodyIn Cancer Therapy,” in Monoclonal Antibodies For Cancer Detection AndTherapy, Baldwin et al., Eds, Academic Press, 1985: Thorpe et al., 1982,Immunol. Rev. 62:119-58; PCT publication WO 89/2624. Any of thesechemistries may be used to link the synthons to an antibody.

Typically, the synthons are linked to the side chains of amino acidresidues of the antibody, including, for example, the primary aminogroup of accessible lysine residues or the sulfhydryl group ofaccessible cysteine residues. Free sulfhydryl groups may be obtained byreducing interchain disulfide bonds. In certain embodiments. LK is alinkage formed with an amino group on the anti-HB7-H3 antibody Ab Incertain embodiments. LK is an amide, thioether, or thiourea In certainembodiments, LK is an amide or thiourea. In certain embodiments. LK is alinkage formed with a sulfhydryl group on the anti-hB7-H3 antibody Ab.In certain embodiments, LK is a thioether. In certain embodiments. LK isan amide, thioether, or thiourea, and m is an integer ranging from 1 to8.

A number of functional groups R^(x) and chemistries useful for linkingsynthons to accessible lysine residues are known, and include by way ofexample and not limitation NHS-esters and isothiocyanates.

A number of functional groups R^(x) and chemistries useful for linkingsynthons to accessible free sulfhydryl groups of cysteine residues areknown, and include by way of example and not limitation haloacetyls andmaleimides.

However, conjugation chemistries are not limited to available side chaingroups. Side chains such as amines may be converted to other usefulgroups, such as hydroxyls, by linking an appropriate small molecule tothe amine. This strategy can be used to increase the number of availablelinking sites on the antibody by conjugating multifunctional smallmolecules to side chains of accessible amino acid residues of theantibody. Functional groups R^(x) suitable for covalently linking thesynthons to these “converted” functional groups are then included in thesynthons.

The antibody may also be engineered to include amino acid residues forconjugation. An approach for engineering antibodies to includenon-genetically encoded amino acid residues useful for conjugating drugsin the context of ADC's is described in Axup et al., 2003, Proc NatlAcad Set 109:16101-16106 and Tian et al., 2014, Proc Natl Acad Sci111:1776-1771 as are chemistries and functional groups useful forlinking synthons to the non-encoded amino acids.

Exemplary synthons useful for making ADCs described herein include, butare not limited to, the following synthons listed below in Table B.

TABLE B Ex- am- Syn- ple thon No. Code Synthon Structure 2.1  CZ

2.2  DH

2.4  EP

2.5  EF

2.6  EG

2.7  EH

2.8  ER

2.9  ES

2.10  EQ

2.11  EU

2.12  EV

2.13  EW

2.14  EX

2.15  EY

2.16  EZ

2.17  FD

2.18  FS

2.19  FI

2.20  FV

2.21  GC

2.22  GB

2.23  FW

2.24  GD

2.25  GK

2.26  GJ

2.27  GW

2.28  HF

2.29  HG

2.30  HP

2.31  HR

2.32  HU

2.33  HT

2.34  HV

2.35  HZ

2.36  IA

2.37  IF

2.38  IG

2.39  IH

2.40  IJ

2.41  IK

2.42  IL

2.43  IM

2.44  IO

2.45  IP

2.46  IS

2.47  IU

2.48  IV

2.49  IZ

2.50  JD

2.51  JF

2.52  JK

2.53  JJ

2.54  JL

2.55  FE

2.56  GG

2.57  GM

2.58  HD

2.59  HS

2.60  HW

2.61  HX

2.62  HY

2.63  IB

2.64  IE

2.65  II

2.66  KY

2.67  IW

2.68  IY

2.69  JA

2.77  FA

2.78  FJ

2.79  FK

2.80  FQ

2.81  FR

2.82  JE

2.83  JM

2.84  LE

2.85  LH

2.86  LJ

2.87  MA

2.88  MD

2.89  MG

2.90  MS

2.91  MR

2.92  MQ

2.93  MZ

2.94  NA

2.95  NB

2.96  NP

2.97  NN

2.98  NO

2.101 OK

2.102 OW

2.103 PC

2.104 PI

2.105 PJ

2.106 PU

2.107 PV

2.108 PW

2.109 QW

2.110 RM

2.111 RR

2.112 SJ

2.113 SM

2.114 SN

2.115 SS

2.116 TA

2.117 TW

2.118 ST

2.119 ZL

2.120 SX

2.121 SW

2.122 TV

2.123 SZ

2.124 ZM

2.125 SV

2.126 SY

2.127 TK

2.128 TR

2.129 TY

2.130 TX

2.131 TZ

2.132 UA

2.133 UJ

2.134 UK

2.135 UU

2.136 UV

2.137 UZ

2.138 VB

2.139 VC

2.140 VS

2.141 VT

2.142 VY

2.143 WI

2.144 WK

2.145 WP

2.146 XD

2.147 XK

2.148 XL

2.149 YJ

2.150 YQ

2.151 YR

2.152 YS

2.153 YY

2.154 YT

2.155 YU

2.156 YV

2.157 YW

2.158 ZB

2.159 ZC

2.160 ZJ

2.161 ZE

2.162 ZS

2.163 ZW

2.164 ZX

2.166 AAA

2.167 AAD

2.168 AAE

2.169 ABG

2.170 ABL

2.171 ABN

2.172 AAF

2.173 ABO

2.174 ABM

2.175 ABU

2.176 ABV

2.177 (con- trol) LB

2.178 (con- trol) WD

2.179 (con- trol) ZZ

2.180 (con- trol) ZT

2.181 (con- trol) XW

2.182 (con- trol) SE

2.183 (con- trol) SR

2.184 (con- trol) YG

2.185 (con- trol) KZ

In certain embodiments, the synthon is selected from the groupconsisting of synthon examples 2.1, 2.2, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9,2.10, 2.11, 2.12, 2.13, 2.14, 2.15, 2.16, 2.17, 2.18, 2.19, 2.20, 2.21,2.22, 2.23, 2.24, 2.25, 2.26, 2.27, 2.28, 2.29, 2.30, 2.31, 2.32, 2.33,2.34, 2.35, 2.36, 2.37, 2.38, 2.39, 2.40, 2.41, 2.42, 2.43, 2.44, 2.45,2.46, 2.47, 2.48, 2.49, 2.50, 2.51, 2.52, 2.53, 2.54, 2.55, 2.56, 2.57,2.58, 2.59, 2.60, 2.61, 2.62, 2.63, 2.64, 2.65, 2.66, 2.67, 2.68, 2.69,2.77, 2.78, 2.79, 2.80, 2.81, 2.82, 2.83, 2.84, 2.85, 2.86, 2.87, 2.88,2.89, 2.90, 2.91, 2.92, 2.93, 2.94, 2.95, 2.96, 2.97, 2.98, 2.101,2.102, 2.103, 2.104, 2.105, 2.106, 2.107, 2.108, 2.109, 2.110, 2.111,2.112, 2.113, 2.114, 2.115, 2.116, 2.117, 2,118, 2.119, 2.120, 2.121,2.122, 2,123, 2,124, 2.125, 2.126, 2.127, 2.128, 2,129, 2.130, 2.131,2.132, 2.133, 2.134, 2.135, 2.136, 2.137, 2.138, 2.139, 2.140, 2.141,2.142, 2.143, 2.144, 2.145, 2.146, 2.147, 2.148, 2.149, 2.150, 2.151,2.152, 2.153, 2.154, 2.155, 2.156, 2.157, 2.158, 2.159, 2.160, 2.161,2.162, 2,163, 2.164, 2.166, 2.167, 2.168, 2.169, 2.170, 2.171, 2.172,2.173, 2.174, 2.175, and 2.176, or a pharmaceutically acceptable saltthereof. The compound names of these synthon are provided below:

-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](3-sulfopropyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-[4-({[2-[2-({3-[(4-{6-[8-(1,3-benazothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethoxy]ethyl}(2-sulfoethyl)carbamoyl]oxy)methyl)phenyl]-N⁵-carbamoyl-L-ornithinamide;-   methyl    6-[4-(3-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]({[4-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-carbamoyl-L-ornithyl}amino)benzyl]oxy}carbonyl)amino}propyl)-1H-1,2,3-triazol-1-yl]-6-deoxy-beta-L-glucopyranoside;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-(4-{[([2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]{3-[1-(beta-D-glucopyranuronosyl)-1H-1,2,3-triazol-4-yl]propyl}carbamoyl)oxy]methyl}phenyl)-N⁵-carbamoyl-L-ornithinamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[(2R)-1-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](methyl)amino}-1-oxo-3-sulfopropan-2-yl]carbamoyl}oxy)methyl]phenyl}-L-alaninamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl][4-(beta-D-glucopyranosyloxy)benzyl]carbamoyl}oxy)methyl]phenyl}-N-carbamoyl-L-ornithinamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[4-(beta-D-allopyranosyloxy)benzyl][2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-phosphonoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-(4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-phosphonoethyl)carbamoyl}oxy)methyl]phenyl)-L-alaninamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](3-phosphonopropyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[(2R)-1-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]amino}-1-oxo-3-sulfopropan-2-yl]carbamoyl}oxy)methyl]phenyl}-L-alaninamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-[4-({[(2-[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethoxy]ethyl}(3-phosphonopropyl)carbamoyl]oxy}methyl)phenyl]-N⁵-carbamoyl-L-ornithinamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-[4-({[{2-[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethoxy]ethyl}(3-phosphonopropyl)carbamoyl]oxy}methyl)phenyl]-L-alaninamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[([2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](3-phosphonopropyl)carbamoyl}oxy)methyl]phenyl-L-alaninamide;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3-(2-{[(2S)-3-carboxy-2-({[(4-{[(2S)-2-([(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino)-3-methylbutanoyl]amino}propanoyl]amino}benzyl)oxy]carbonyl}amino)propanoyl](methyl)amino}eth    oxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylic    acid;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl][4-(beta-D-glucopyranuronosyloxy)benzyl]carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[(1-[2-({3-[(4-{6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]-2-carboxypyridin-3-yl)-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-phosphonoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-carbamoyl-N-{4-[({[2-({3-[(4-{2-carboxy-6-[8-([1,3]thiazolo[5,4-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methy]phenyl}-L-ornithinamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[(2R)-1-[2-({3-[(4-{6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-v]-2-carboxypyridin-3-yl)-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](methy)amino}-1-oxo-3-sulfopropan-2-yl]carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[(2R)-1-{[2-({3-[(4-{6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](methyl)amino}-1-oxo-3-sulfopropan-2-yl]carbamoyl}oxy)methyl]phenyl}-L-alaninamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-carbamoyl-N-{4-[({1-[2-({3-[(4-{2-carboxy-6-[8-([1,3]thiazolo[4,5-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-L-ornithinamide-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N-carbamoyl-L-ornithinamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-carboxyethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-([4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-carboxyethyl)carbamoyl}oxy)methyl]phenyl}-L-alaninamide;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(l-{[3-(2-{([(2R)-3-carboxy-2-({[(4-{[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl)oxy]carbonyl}amino)propanoyl](methyl)amino}eth    oxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylic    acid;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl][1-(carboxymethyl)piperidin-4-yl]carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide;-   (S)-6-((2-((3-((4-(6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-2-carboxypyridin-3-yl)-5-methyl-1H-pyrazol-1-yl)methyl)-5,7-dimethyladamantan-1-yl)oxy)ethyl)(methyl)amino)-5-((((4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)amino)-N,N,N-trimethyl-6-oxohexan-1-aminium    salt;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-L-alaninamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-[4-({[(4-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)amino}piperidin-1-yl)carbonyl]oxy}methyl)phenyl]-N⁵-carbamoyl-L-ornithinamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-5-(3-phosphonopropoxy)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](methyl)carbamoyl}oxy)methyl]phenyl}-N-carbamoyl-L-ornithinamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-[4-({[(4-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](3-phosphonopropyl)amino}piperidin-1-yl)carbonyl]oxy}methyl)phenyl]-N⁵-carbamoyl-L-ornithinamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](3-phosphonopropyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-[4-({[(4-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-carboxyethyl)amino}piperidin-1-yl)carbonyl]oxy}methyl)phenyl]-N⁵-carbamoyl-L-ornithinamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-carbamoyl-N-{4-[({[2-({3-[(4-{2-carboxy-6-[8-([1,3]thiazolo[4,5-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(H)-yl]pyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](3-phosphonopropyl)carbamoyl}oxy)methyl]phenyl}-L-ornithinamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N³-carbamoyl-N-{4-[({[2-({3-[(4-{2-carboxy-6-[8-([1,3]thiazolo[5,4-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](3-phosphonopropyl)carbamoyl}oxy)methyl]phenyl}-L-ornithinamide;-   N-{6-[(chloroacetyl)amino]hexanoyl}-L-valyl-N⁵-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-L-alaninamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-5-(carboxymethoxy)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](methyl)carbamoyl}oxy)methyl]phenyl}-N³-carbamoyl-L-ornithinamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-[4-({[(2-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)amino}ethyl)(2-carboxyethyl)carbamoyl]oxy}methyl)phenyl]-N⁵-carbamoyl-L-ornithinamide;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-[1-({3-[2-({(2S)-2-[{1[(4-{1[(2S)-5-(carbamoylamino)-2-{1[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}pentanoyl]amino}benzyl)oxy]carbonyl}(2-carboxyethyl)amino]-3-carboxypropanoyl}amino)ethoxy]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3-(2-{[(2S)-2-({[(4-{1[(2S)-5-(carbamoylamino)-2-{1[(2S)-2-{1-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}pentanoyl]amino}benzyl)oxy]carbonyl}amino)-3-carboxypropanoyl](2-sulfoethyl)amino}ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylic    acid;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-[4-({[(4-{1-[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](3-carboxypropyl)amino}piperidin-1-yl)carbonyl]oxy}methyl)phenyl]-N′-carbamoyl-L-ornithinamide;-   4-[(1E)-3-({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-5-(carboxymethoxy)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](methyl)carbamoyl}oxy)prop-1-en-1-yl]-2-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-beta-alanyl}amino)phenyl    beta-D-glucopyranosiduronic acid;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1,]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-[4-({1[(2-{[8-(1,3-benzothiazol-2-ylcarbamoyl)-2-(6-carboxy-5-{1-[(3,5-dimethyl-7-{2-[methyl(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methy]-5-methyl-1H-pyrazol-4-yl}pyridin-2-yl)-1,2,3,4-tetrahydroisoquinolin-5-yl]oxy}ethyl)carbamoyl]oxy}methyl)phenyl]-N⁵-carbamoyl-L-ornithinamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-[4-({[(2-{[8-(1,3-benzothiazol-2-ylcarbamoyl)-2-(6-carboxy-5-{1-[(3,5-dimethyl-7-{2-[methyl(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridin-2-yl)-1,2,3,4-tetrahydroisoquinolin-5-yl]oxy}ethyl)(2-sulfoethyl)carbamoyl]oxy}methyl)phenyl]-N-carbamoyl-L-ornithinamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-[4-({1[(2-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)amino}ethyl)(2-sulfoethyl)carbamoyl]oxy}methyl)phenyl]-N⁵-carbamoyl-L-ornithinamide;-   N-{6-[(chloroacetyl)amino]hexanoyl}-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({1-[2-({3-[(4-{6-[4-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydro-2H-1,4-benzoxazin-6-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N-carbamoyl-L-ornithinamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-[4-({[(2-{[8-(1,3-benzothiazol-2-ylcarbamoyl)-2-(6-carboxy-5-{1-[(3,5-dimethyl-7-{2-[methyl(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridin-2-yl)-1,2,3,4-tetrahydroisoquinolin-5-y]oxy}ethyl)(2-carboxyethyl)carbamoyl]oxy}methyl)phenyl]-N⁵-carbamoyl-L-ornithinamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({1-[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-5-(3-sulfopropoxy)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](methyl)carbamoyl}oxy)methyl]phenyl}-N-carbamoyl-L-ornithinamide;-   N-[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide;-   N-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3-(2-{[(2S)-2-({[(4-{[(2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl]oxy}-3-[(3-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}propanoyl)amino]benzyl)oxy]carbonyl}amino)-3-sulfopropanoyl](methyl)amino}ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylic    acid;-   4-[(1E)-3-({1-[2-({3-[(4-{2-carboxy-6-[8-([1,3]thiazolo[5,4-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)prop-1-en-1-yl]-2-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-beta-alanyl}amino)phenyl    beta-D-glucopyranosiduronic acid;-   4-[(1E)-3-({[2-({(3-[(4-{2-carboxy-6-[8-([1,3]thiazolo[4,5-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)prop-1-en-1-yl]-2-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-beta-alanyl}amino)phenyl    beta-D-glucopyranosiduronic acid;-   4-[(1E)-3-({([2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoy)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)prop-1-en-1-yl]-2-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-beta-alanyl}amino)phenyl    beta-D-glucopyranosiduronic acid;-   4-[(1E)-3-({[2-({(3-[(4-{2-carboxy-6-[8-([1,3]thiazolo[5,4-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](3-phosphonopropyl)carbamoyl}oxy)prop-1-en-1-yl]-2-({(N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-beta-alanyl}amino)penyl    beta-D-glucopyranosiduronic acid;-   4-[(1E)-3-({([2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-5-(3-phosphonopropoxy)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](methyl)carbamoyl}oxy)prop-1-en-1-yl]-2-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-beta-alanyl}amino)phenyl    beta-D-glucopyranosiduronic acid;-   4-[(1E)-3-({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethy](3-phosphonopropyl)carbamoyl}oxy)prop-1-en-1-yl]-2-((N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-beta-alanyl)amino)phenyl    beta-D-glucopyranosiduronic acid;-   4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-3-[2-(2-{[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]amino}ethoxy)ethoxy]phenyl    beta-D-glucopyranosiduronic acid;-   4-[(1E)-3-({[2-({3-[(4-{(2-carboxy-6-[8-([1,3]thiazolo[4,5-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1³.]dec-1-yl}oxy)ethyl](3-phosphonopropyl)carbamoyl}oxy)prop-1-en-1-yl]-2-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-beta-alanyl}amino)phenyl    beta-D-glucopyranosiduronic acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[(2-carboxyethyl)({[(2E)-3-(4-{[(2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl]oxy}-3-[(3-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}propanoyl)amino]phenyl)prop-2-en-1-yl]oxy}carbonyl}amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[(2-carboxyethyl){[(4-{[(2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl]oxy}-2-[2<2-{([3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]amino}ethoxy)ethoxy]benzyl)oxy]carbonyl}amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   N-[6-(ethenylsulfonyl)hexanoyl]-L-valyl-N-(4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N-carbamoyl-L-ornithinamide;-   4-[(1E)-3-{[(4-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)l]dec-1-yl}oxy)ethyl](3-phosphonopropyl)amino}piperidin-1-yl)carbonyl]oxy}prop-1-en-1-yl]-2-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-beta-alanyl}amino)phenyl    beta-D-glucopyranosiduronic acid;-   4-[(1E)-3-{[(4-f[2-({3-[(4-{2-carboxy-6-[8-([1,3]thiazolo[4,5-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](3-phosphonopropyl)amino}piperidin-1-yl)carbonyl]oxy    prop-1-en-1-yl]-2-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-beta-alanyl}amino)phenyl    beta-D-glucopyranosiduronic acid;-   4-[(1E)-3-([2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-2-carboxypyridin-3-yl)-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)prop-1-en-1-yl]-2-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-beta-alanyl}amino)phenyl    beta-D-glucopyranosiduronic acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-[1-({3-[2-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-3-sulfo-L-alanyl}amino)ethoxy]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-[1-({3-[2(2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-y)hexanoyl](2-sulfoethyl)amino}ethoxy)ethoxy]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3-(2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl](2-sulfoethyl)amino}ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo-22-(2-sulfoethyl)-3,6,9,12,15,18-hexaoxa-22-azatetracosan-24-yl]oxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{([1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo-22-(2-sulfoethyl)-3,6,9,12,15,18,25-heptaoxa-22-azaheptacosan-27-yl]oxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3-(2-{[6-(ethenylsulfonyl)hexanoyl](2-sulfoethyl)amino}ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(H)-yl]-3-{1-[(3-{2-[{6-[(chloroacetyl)amino]hexanoyl}(2-sulfoethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-(4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](3-carboxypropyl)carbamoyl)oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide;-   N-{(6-[(bromoacetyl)amino]hexanoyl}-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N-carbamoyl-L-ornithinamide;-   4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](3-carboxypropyl)carbamoyl}oxy)methyl]-3-[2-(2-{[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]amino}ethoxy)ethoxy]phenyl    beta-D-glucopyranosiduronic acid;-   4-({[(4-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](3-carboxypropyl)amino}piperidin-1-yl)carbonyl]oxy}methyl)-3-[2-(2-{[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]amino}ethoxy)ethoxy]phenyl    beta-D-glucopyranosiduronic acid;-   4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](3-sulfopropyl)carbamoyl}oxy)methyl]-3-[2-(2-{[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]amino}ethoxy)ethoxy]phenyl    beta-D-glucopyranosiduronic acid;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-[4-({[(3-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)amino}azetidin-1-yl)carbonyl]oxy}methyl)phenyl]-N⁵-carbamoyl-L-ornithinamide;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{[26-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-8,24-dioxo-3-(2-sulfoethyl)-11,14,17,20-tetraoxa-3,7,23-triazahexacos-1-yl]oxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-[4-({[(3-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)amino}propyl)carbamoy]oxy}methyl)phenyl]-N⁵-carbamoyl-L-ornithinamide;-   N-{6-[(iodoacetyl)amino]hexanoyl}-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N-carbamoyl-L-ornithinamide;-   N-{6-[(ethenylsulfonyl)amino]hexanoyl}-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide;-   N-{6-[(ethenylsulfonyl}amino]hexanoyl)-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[(3-{[6-(ethenylsulfonyl)hexanoyl]amino}propyl)(2-sulfoethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   N-[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(H)-yl]-3-{1-[(3-{2-[(2-carboxyethyl)){[(2-{[(2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl]oxy}-4-[2-(2-{[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]amino}ethoxy)ethoxy]benzyl)oxy]carbonyl}amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-3-sulfo-L-alany-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-carboxyethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{[(43S,46S)-43-({[(4-{[(2S)-2-([(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl)oxy]carbonyl}amino)-46-methyl-37,44,47-trioxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-38,45,48-triazapentacontan-50-yl]oxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)-]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylic    acid;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-carboxyethyl)carbamoyl}oxy)methyl]phenyl}-N-carbamoyl-L-ornithinamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-carboxyethyl)carbamoyl}oxy)methyl]phenyl}-N-carbamoyl-L-ornithinamide;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-3-{1-[(3-{2-[(2-carboxyethyl){[(2-{[(2R,3S,4R,5R,6R)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl]oxy}-4-[2-(2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}ethoxy)ethoxy]benzyl)oxy]carbonyl}amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[5-(1,3-benzothiazol-2-ylcarbamoyl)quinolin-3-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)l]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N-carbamoyl-L-ornithinamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[4-(1,3-benzothiazol-2-ylcarbamoyl)quinolin-6-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-carboxyethyl)carbamoyl}oxy)methyl]phenyl}-N-carbamoyl-L-ornithinamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[4-(1,3-benzothiazol-2-ylcarbamoyl)quinolin-6-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[5-(1,3-benzothiazol-2-ylcarbamoyl)quinolin-3-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-carboxyethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N-carbamoyl-L-ornithinamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[7-(1,3-benzothiazol-2-ylcarbamoyl)-1H-indol-2-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-[4-({[{3-[8-(1,3-benzothiazol-2-ylcarbamoyl)-2-(6-carboxy-5-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridin-2-yl)-1,2,3,4-tetrahydroisoquinolin-6-yl]propyl}(methyl)carbamoyl]oxy}methyl)phenyl]-N⁵-carbamoyl-L-ornithinamide;-   N-(6-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}hexanoyl)-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl][3-(beta-L-glucopyranuronosyloxy)propyl]carbamoyl}oxy)methyl]phenyl}-N-carbamoyl-L-ornithinamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[4-(1,3-benzothiazol-2-ylcarbamoyl)isoquinolin-6-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-alpha-glutamyl-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide;-   N-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L-alpha-glutamyl-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide;-   1-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]({[4-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-D-valyl-N-carbamoyl-D-ornithyl}amino)benzyl]oxy}carbonyl)amino}-1,2-dideoxy-D-arabino-hexitol;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[4-(1,3-benzothiazol-2-ylcarbamoyl)-2-oxidoisoquinolin-6-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](methyl)carbamoyl}oxy)methyl]phenyl}-N⁴-carbamoyl-L-ornithinamide;-   N-({(3S,5S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-5-[(2-sulfoethoxy)methyl]pyrrolidin-1-yl}acetyl)-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide;-   N-{(2S)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-[4-(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yloxy)phenyl]propanoyl}-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N-carbamoyl-L-ornithinamide;-   N-({(3S,5S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-5-[(2-sulfoethoxy)methyl]pyrrolidin-1-yl}acetyl)-L-valyl-N-{4-[({[2-(3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl)oxy)methyl]phenyl}-N-carbamoyl-L-ornithinamide;-   N-{(2S)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-[4-(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yloxy)phenyl]propanoyl}-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N-carbamoyl-L-ornithinamide;-   (6S)-2,6-anhydro-6-(2-{2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-5-({N-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L-valyl-L-alanyl}amino)phenyl}ethyl)-L-gulonic    acid;-   3-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]({[4-(4-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}butyl)-2-(beta-D-glucopyranuronosyloxy)benzyl]oxy}carbonyl)amino}propyl    beta-D-glucopyranosiduronic acid;-   N-{[(3S,5S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-(methoxymethyl)-2-oxopyrrolidin-1-yl]acetyl}-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide;-   (6S)-2,6-anhydro-6-(2-{2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-5-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-L-alanyl}amino)phenyl}ethyl)-L-gulonic    acid;-   2-[({([2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-5-(4-{[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]amino}butyl)phenyl    beta-D-glucopyranosiduronic acid;-   2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)-]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-5-[4-({(2S)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-[4-(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yloxy)phenyl]propanoyl}amino)butyl]phenyl    beta-D-glucopyranosiduronic acid;-   (6S)-2,6-anhydro-6-(2-(2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-5-[(N-{(2S)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-[4-(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yloxy)phenyl]propanoyl}-L-valyl-L-alanyl)amino]phenyl}ethyl)-L-gulonic    acid;-   6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(H)-yl)-3-(1-((3-(2-((((2-(2-((2S,3R,4R,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)ethyl)-4-((S)-2-((S)-2-(2-((3S,5S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-5-((2-sulfoethoxy)methyl)pyrrolidin-1-yl)acetamido)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)(2-sulfoethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinic    acid;-   6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-(2-((((2-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-4-(4-(2-((3S,5S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-5-((2-sulfoethoxy)methyl)pyrrolidin-1-yl)acetamido)butyl)benzyl)oxy)carbonyl)(2-sulfoethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinic    acid;-   2-[({[2-{3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-5-(4-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}butyl)phenyl    beta-D-glucopyranosiduronic acid;-   2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-5-(4-([(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino)butyl)phenyl    beta-D-glucopyranosiduronic acid;-   2-[({[2-(13-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl)oxy)methyl]-5-[4-({(2S)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-[4-(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yloxy)phenyl]propanoyl}amino)butyl]phenyl    beta-D-glucopyranosiduronic acid;-   N-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-3-(4-carboxybutyl)phenyl}-L-alaninamide;-   2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-5-(3-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}propyl)phenyl    beta-D-glucopyranosiduronic acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(H)-yl]-3-{1-[(3-{2-[({[2-{[(2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl]oxy}-4-(4-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}butyl)benzyl]oxy}carbonyl)(3-([1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino}-3-oxopropyl)amino]ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-(8-(benzo[d]thiazol-2-ylcarbamoyl)naphthalen-2-yl)-3-(1-((3-(2-((((2-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-4-(4-(2-((3S,5S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-5-((2-sulfoethoxy)methyl)pyrrolidin-1-yl)acetamido)butyl)benzyl)oxy)carbonyl)(2-sulfoethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinic    acid;-   2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl][3-hydroxy-2-(hydroxymethyl)propyl]carbamoyl}oxy)methyl]-5-(3-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}propyl)phenyl    beta-D-glucopyranosiduronic acid;-   N-({(3S,5S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-5-[(2-sulfoethoxy)methyl]pyrrolidin-1-yl}acetyl)-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methy]-3-(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50-heptadecaoxatripentacont-52-yn-53-yl)phenyl}-L-alaninamide;-   N-({(3S,5S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-5-[(2-sulfoethoxy)methyl]pyrrolidin-1-yl}acetyl)-L-valy-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-3-(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50-heptadecaoxatripentacontan-53-yl)phenyl}-L-alaninamide;-   2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl][(3S)-3,4-dihydroxybutyl]carbamoyl}oxy)methyl]-5-(3-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}propyl)phenyl    beta-D-glucopyranosiduronic acid;-   1-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1⁷′]dec-1-yl}oxy)ethyl]({[4-(4-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}butyl)-2-(beta-D-glucopyranuronosyloxy)benzyl]oxy}carbonyl)amino}-1,2-dideoxy-D-arabino-hexitol;-   1-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]({[4-(4-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}butyl)-2-(beta-D-glucopyranuronosyloxy)benzyl]oxy}carbonyl)amino}-1,2-dideoxy-D-erythro-pentitol;-   N-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L-valyl-N-{4-[({[2-({3-[(4-(6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-v]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-3-[27-(2,5,8,11,14,17,20,23-octaoxahexacosan-26-yl)-2,5,8,11,14,17,20,23-octaoxa-27-azatriacontan-30-yl]phenyl}-L-alaninamide;-   (6S)-2,6-anhydro-6-(2-{2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-5-({N-[(2S)-3-[3,4-bis(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yloxy)phenyl]-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]-L-valyl-L-alanyl}amino)phenyl}ethyl)-L-gulonic    acid;-   N-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-N-(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yl)-beta-alanyl-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-3-(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50-heptadecaoxatripentacontan-53-yl)phenyl}-L-alaninamide;-   N-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-N-(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yl)-beta-alanyl-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide;-   N-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L-valyl-N-{4-[({1-[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-3-[27-(2,5,8,11,14,17,20,23-octaoxahexacosan-26-yl)-2,5,8,11,14,17,20,23-octaoxa-27-azatriacontan-30-yl]phenyl}-L-alaninamide;-   N-{(3S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-[1-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-yl)-1H-1,2,3-triazol-4-yl]propanoyl}-L-valyl-N-{4-[({[2-(13-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl)-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide;-   N-{(3R)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-[1-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-yl)-1H-1,2,3-triazol-4-yl]propanoyl}-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N-carbamoyl-L-ornithinamide;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-[1-({3-[2-({[(2-{2-[(2S,3R,4R,5S,6S)-6-carboxy-3,4,5-trihydroxy    tetrahydro-2H-pyran-2-yl]ethyl}-4-{[(2S)-2-{[(2S)-2-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl)oxy]carbonyl}[(3R,4S,5R)-3,4,5,6-tetrahydroxyhexyl]amino)ethoxy]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-[1-({3-[2-({[(2-{2-[(2S,3R,4R,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl]ethyl)-4-{1[(2S)-2-({(2S)-2-[(((3S,5S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-5-[(2-sulfoethoxy)methyl]pyrrolidin-1-yl}acetyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl)oxy]carbonyl}[(3R,4S,5R)-3,4,5,6-tetrahydroxyhexyl]amino)ethoxy]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylic    acid;-   (6S)-2,6-anhydro-6-(2-{2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-5-((N-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-N-(2.5.8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yl)-beta-alanyl-L-valyl-L-alanyl}amino)phenyl}ethyl)-L-gulonic    acid;-   (6S)-2,6-anhydro-6-(2-{2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl)oxy}methy]-5-[(N-{2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-[1-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-yl)-1H-1,2,3-triazol-4-yl]propanoyl}-L-valyl-L-alanyl)amino]phenyl}ethyl)-L-gulonic    acid;-   (6S)-2,6-anhydro-6-(2-{2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-5-[(N-{(3S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-[1-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-yl)-1H-1,2,3-triazol-4-yl]propanoyl}-L-valyl-L-alanyl)amino]phenyl}ethyl)-L-gulonic    acid;-   (6S)-2,6-anhydro-6-(2-{2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-y)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-5-[(N-{(3R)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-[1-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-yl)-1H-1,2,3-triazol-4-yl]propanoyl}-L-valyl-L-alanyl)amino]phenyl}ethyl)-L-gulonic    acid;-   (6S)-2,6-anhydro-6-(2-{2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-5-[(N-{(3S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-[1-(3-sulfopropyl)-1H-1,2,3-triazol-4-yl]propanoyl}-L-valyl-L-alanyl)amino]phenyl}ethyl)-L-gulonic    acid;-   (6S)-2,6-anhydro-6-(2-{2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-5-[(N-{(3R)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-[1-(3-sulfopropyl)-1H-1,2,3-triazol-4-yl]propanoyl}-L-valyl-L-alanyl)amino]phenyl}ethyl)-L-gulonic    acid;-   (6S)-2,6-anhydro-6-(2-{2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-5-({N-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-N-[2-(2-sulfoethoxy)ethyl]-beta-alanyl-L-valyl-L-alanyl}amino)phenyl}ethyl)-L-gulonic    acid;-   6-{8-[(1,3-benzothiazol-2-yl)carbamoyl]-3,4-dihydroisoquinolin-2(1H)-yl}-3-[1-({3-[2-({[(2-{2-[(2S,3R,4R,5S,6S)-6-carboxy-3,4,5-trihydroxyoxan-2-yl]ethyl}-4-{[(2S)-2-{[(2S)-2-{[(2S)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-{4-[(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yl)oxy]phenyl}propanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}phenyl)methoxy]carbonyl}[(3R,4S,5R)-3,4,5,6-tetrahydroxyhexyl]amino)ethoxy]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]decan-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylic    acid;-   4-{[({2-[(3-{1-[4-(6-{8-[(1,3-benzothiazol-2-yl)carbamoyl]-3,4-dihydroisoquinolin-2(1H)-yl}-2-carboxypyridin-3-yl)-5-methyl-1H-pyrazol-1-yl]methyl}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]decan-1-yl)oxy]ethyl}[(3S)-3,4-dihydroxybutyl]carbamoyl)oxy]methyl}-3-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido]ethoxy}ethoxy)phenyl    beta-D-glucopyranosiduronic acid;-   2,6-anhydro-8-[2-({[{2-[(3-{-[4-(6-{8-[(1,3-benzothiazol-2-yl)carbamoyl]-3,4-dihydroisoquinolin-2(1H)-yl}-2-carboxypyridin-3-yl)-5-methyl-1H-pyrazol-1-yl]methyl}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]decan-1-yl)oxy]ethyl}(2-sulfoethyl)carbamoyl]oxy}methyl)-5-{([(79S,82S)-74-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-82-methyl-77,80,83-trioxo-79-(propan-2-yl)-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-74,78,81-triazatrioctacontan-83-yl]amino}phenyl]-7,8-dideoxy-L-glycero-L-gulo-octonic    acid;-   6-{8-[(1,3-benzothiazol-2-yl)carbamoyl]-3,4-dihydroisoquinolin-2(1H)-yl}-3-{1-[(3-{2-[{1[(4-{[(2S,5S)-2-[3-(carbamoylamino)propyl]-10-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-4,7-dioxo-5-(propan-2-yl)-15-sulfo-13-oxa-3,6,10-triazapentadecanan-1-oyl]amino}phenyl)methoxy]carbonyl}(2-sulfoethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]decan-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-(2-((((2-(2-((2S,3R,4R,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)ethyl)-4-((S)-2-((S)-2-(2-((3S,5S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-5-((2-sulfoethoxy)methyl)pyrrolidin-1-y)acetamido)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)((S)-3,4-dihydroxybutyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinic    acid;-   2,6-anhydro-8-(2-{[({2-[(3-{[4-(6-{8-[(1,3-benzothiazol-2-yl)carbamoyl]-3,4-dihydroisoquinolin-2(1H)-yl}-2-carboxypyridin-3-yl)-5-methyl-1H-pyrazol-1-yl]methyl}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]decan-1-yl)oxy]ethyl}[(3S)-3,4-dihydroxybutyl]carbamoyl)oxy]methyl}-5-{1[(2S)-2-({(2S)-2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido]-3-methylbutanoyl}amino)propanoyl]amino}phenyl)-7,8-dideoxy-L-glycero-L-gulo-octonic    acid;-   2-{[({2-[(3-{[4-(6-{8-[(1,3-benzothiazol-2-yl)carbamoyl]-3,4-dihydroisoquinolin-2(1H)-yl}-2-carboxypyridin-3-yl)-5-methyl-1H-pyrazol-1-yl]methyl}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]decan-1-yl)oxy]ethyl}[(3S)-3,4-dihydroxybutyl]carbamoyl)oxy]methyl}-5-{4-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido]butyl}phenyl    beta-D-glucopyranosiduronic acid;-   6-{8-[(1,3-benzothiazol-2-yl)carbamoyl]-3,4-dihydroisoquinolin-2(1H)-yl}-3-{1-[(3-{2-[{[(4-{[(2S)-5-(carbamoylamino)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}pentanoyl]amino}phenyl)methoxy]carbonyl}(2-sulfoethyl)amino]acetamido}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]decan-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-y)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}sulfanyl)ethyl](2-sulfoethyl)carbamoyl})oxy)methyl    1]phenyl}-N⁵-carbamoyl-L-ornithinamide;-   N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-[4-({[(3-{3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}propyl)(2-sulfoethyl)carbamoyl]oxy}methyl)phenyl]-N⁵-carbamoyl-L-ornithinamide;-   2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl][(3S)-3,4-dihydroxybutyl]carbamoyl}oxy)methyl]-5-{4-[({(3S,5S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-5-[(2-sulfoethoxy)methyl]pyrrolidin-1-yl}acetyl)amino]butyl}phenyl    beta-D-glucopyranosiduronic acid;-   2,6-anhydro-8-[2-({[{2-[(3-([4-(6-{8-[(1,3-benzothiazol-2-yl)carbamoyl]-3,4-dihydroisoquinolin-2(1H)-yl}-2-carboxypyridin-3-yl)-5-methyl-1H-pyrazol-1-yl]methyl}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]decan-1-yl)oxy]ethyl}(2-sulfoethyl)carbamoyl]oxy)methyl)-5-([N-({(3R,5S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-5-[(2-sulfoethoxy)methyl]pyrrolidin-1-yl}acetyl)-L-valyl-L-alanyl]amino)phenyl]-7,8-dideoxy-L-glycero-L-gulo-octonic    acid;-   2,6-anhydro-8-{2-({[{2-[(3-{[4-(6-{8-[(1,3-benzothiazol-2-yl)carbamoyl]-3,4-dihydroisoquinolin-2(1H)-yl}-2-carboxypyridin-3-yl)-5-methyl-1H-pyrazol-1-yl]methyl}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]decan-1-yl)oxy]ethyl}(2-sulfoethyl)carbamoyl]oxy}methyl)-5-[(N-{[(3R,5S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-5-(41-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38-tridecaoxa-42-azatritetracontan-43-yl)pyrrolidin-1-yl]acetyl}-L-valyl-L-alanyl)amino]phenyl}-7,8-dideoxy-L-glycero-L-gulo-octonic    acid;-   (6S)-2,6-anhydro-6-(2-{2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl][(3S)-3,4-dihydroxybutyl]carbamoyl}oxy)methyl]-5-({N-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-N-(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yl)-b-alanyl-L-valyl-L-alanyl}amino)phenyl}ethyl)-L-gulonic    acid; and-   (6S)-2,6-anhydro-6-(2-{2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl][(3S)-3,4-dihydroxybutyl]carbamoyl}oxy)methyl]-5-({N-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-N-[2-(2-sulfoethoxy)ethyl]-b-alanyl-L-valyl-L-alanyl}amino)phenyl}ethyl)-L-gulonic    acid.

In certain embodiments, the ADC, or a pharmaceutically acceptable saltthereof.

D is the Bcl-xL inhibitor selected from the group consisting of thefollowing compounds modified in that the hydrogen corresponding to the 4position of structural formula (IIa), (IIb), (IIc), or (IId) is notpresent, forming a monoradical:

W2.01, W2.02, W2,03, W2.04, W2.05, W2.06, W2,07, W2,08, W2.09, W2.10,W2.11, W2.12, W2.13, W2.14, W2.15, W2.16, W2.17, W2.18, W2.19, W2.20,W2.21, W2.22, W2.23, W2.24, W2.25, W2.26, W2,27, W2.28, W2.29, W2.30,W2,31, W2.32, W2.33, W2.34, W2.35, W2.36, W2.37, W2.38, W2.39, W2.40,W2.41, W2.42, W2.43, W2.44, W2.45, W2.46, W2.47, W2.48, W2.49, W2.50,W2.51, W2.52, W2,53, W2.54, W2.55, W2.56, W2.57, W2,58, W2.59, W2.60,W2.61, W2.62, W2.63, W2.64, W2.65, W2.66, W2.67, W2.68, W2.69, W2.70,W2.71, W2.72, W2.73, W2.74, W2.75, W2.76, W2.77, W2.78, W2.79, W2.80,W2.81, W2.82, W2.83, W2,84, W2.85, W2.86, W2.87, W2.88, W2,89, W2.90,and W2.91, and a pharmaceutically acceptable salt thereof;

L is selected from the group consisting of linkers IVa.1-IVA.8,IVb.1-IVb.19, IVc.1-IVc.7, IVd.1-IVd.4, Va.1-Va.12, Vb.1-Vb.10,Vc.1-Vc.11, Vd.1-Vd.6, Ve.1-Ve.2, VIa.1, VIc.1-VIc.2, VId.1-VId.4,VIIa.1-VIIa.4, VIIb.1-VIIb.8, VIIc.1-VIIc.6, wherein each linker hasreacted with the antibody, Ab, forming a covalent attachment;

LK is thioether and

m is an integer ranging from 1 to 8.

In certain embodiments, the ADC, or a pharmaceutically acceptable saltthereof.

D is the Bcl-xL inhibitor selected from the group consisting of thefollowing compounds modified in that the hydrogen corresponding to the 4position of structural formula (IIa), (IIb), (IIc), or (IId) is notpresent, forming a monoradical:

-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl-}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[(2-carboxyethyl)    amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3,3,1,1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic    acid;-   1-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]amino}-1,2-dideoxy-D-arabino-hexitol;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3-(2-{[3-hydroxy-2-(hydroxymethyl)propyl]amino}ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylic    acid;-   6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3-(2{[(3S)-3,4-dihydroxybutyl]amino}ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylic    acid;

and pharmaceutically acceptable salts thereof;

L is selected from the group consisting of linkers IVb.2, IVc.5, IVc.6,IVc.7, IVd.4, Vb.9, Vc.11, VIIa.1, VIIa.3, VIIc.1, VIIc.4, and VIIc.5 ineither closed or open limns and a pharmaceutically acceptable saltthereof;

LK is thioether; and

m is an integer ranging from 2 to 4.

To form an ADC, the maleimide ring of a synthon (for example, thesynthons listed in Table B) may react with an antibody Ab, forming acovalent attachment as either a succinimide (closed form) or succinamide(open form), Similarly, other functional groups, e.g. acetyl halide orvinyl sulfone may react with an antibody, Ab, forming a covalentattachment.

In certain embodiments, the ADC, or a pharmaceutically acceptable saltthereof, is selected from the group consisting of AbA-CZ, AbA-TX,AbA-TV, AbA-YY, AbA-AAA, AbA-AAD, AbB-CZ, AbB-TX, AbB-TV, AbB-YY,AbB-AAD, AhG-CZ, AbG-TX, AbG-TV, AbG-YY, AbG-AAA, AbG-AAD, AbK-CZ,AbK-TX, AbK-TV, AbK-YY, AbK-AAA, AbK-AAD, wherein CZ, TX, TV, YY. AAA,and AAD are synthons disclosed in Table B, and wherein the conjugatedsynthons are either in open or closed form.

In one embodiment, the ADC, or a pharmaceutically acceptable saltthereof, is:

wherein in is 2, Ab is either an anti-hB7-H3 antibody, wherein theanti-hB7-H3 antibody comprises a heavy chain CDR3 domain comprising theamino acid sequence set forth in SEQ ID NO: 35, a heavy chain CDR2domain comprising the amino acid sequence set forth in SEQ ID NO: 34,and a heavy chain CDR1 domain comprising the amino acid sequence setforth in SEQ ID NO: 33; and a light chain CDR3 domain comprising theamino acid sequence set forth in SEQ ID NO: 39, a light chain CDR2domain comprising the amino acid sequence set forth in SEQ ID NO: 38,and a light chain CDR1 domain comprising the amino acid sequence setforth in SEQ ID NO: 37; or an anti-hB7-H3 antibody, wherein theanti-hB7H3 antibody comprises a heavy chain variable region comprisingthe amino acid sequence set forth in SEQ ID NO: 147, and a light chainvariable region comprising the amino acid sequence set forth in SEQ IDNO: 144; or an anti-hB7-H3 antibody, wherein the anti-hB7-H3 antibodycomprises a heavy chain comprising the amino acid sequence set forth inSEQ ID NO: 168, and a light chain comprising the amino acid sequence setforth in SEQ ID NO: 169.

In one embodiment, the ADC, or a pharmaceutically acceptable saltthereof, is:

wherein m is 2, Ab is either an anti-hB7-H3 antibody, wherein theanti-hB7-H3 antibody comprises a heavy chain CDR3 domain comprising theamino acid sequence set forth in SEQ ID NO: 12, a heavy chain CDR2domain comprising the amino acid sequence set forth in SEQ ID NO: 140,and a heavy chain CDR1 domain comprising the amino acid sequence setforth in SEQ ID NO: 10; and a light chain CDR3 domain comprising theamino acid sequence set forth in SEQ ID NO: 15, a light chain CDR2domain comprising the amino acid sequence set forth in SEQ ID NO: 7, anda light chain CDR1 domain comprising the amino acid sequence set forthin SEQ ID NO: 136; or an anti-hB7-H3 antibody, wherein the anti-hB7H3antibody comprises a heavy chain variable region comprising the aminoacid sequence set forth in SEQ ID NO: 139, and a light chain variableregion comprising the amino acid sequence set forth in SEQ ID NO: 135;or an anti-hB7-H3 antibody, wherein the anti-hB7-H3 antibody comprises aheavy chain comprising the amino acid sequence set forth in SEQ ID NO:170, and a light chain comprising the amino acid sequence set forth inSEQ ID NO: 171.

In one embodiment, the ADC, or a pharmaceutically acceptable saltthereof, is:

wherein m is 2, Ab is either an anti-hB7-H3 antibody, wherein theanti-hB7-H3 antibody comprises a heavy chain CDR3 domain comprising theamino acid sequence set forth in SEQ ID NO: 35, a heavy chain CDR2domain comprising the amino acid sequence set forth in SEQ ID NO: 34,and a heavy chain CDR1 domain comprising the amino acid sequence setforth in SEQ ID NO: 33; and a light chain CDR3 domain comprising theamino acid sequence set forth in SEQ ID NO: 39, a light chain CDR2domain comprising the amino acid sequence set forth in SEQ ID NO: 38,and a light chain CDR1 domain comprising the amino acid sequence setforth in SEQ ID NO: 37; or an anti-hB7-H3 antibody, wherein theanti-hB7H3 antibody comprises a heavy chain variable region comprisingthe amino acid sequence set forth in SEQ ID NO: 147, and a light chainvariable region comprising the amino acid sequence set forth in SEQ IDNO: 144; or an anti-hB7-H3 antibody, wherein the anti-hB7-H3 antibodycomprises a heavy chain comprising the amino acid sequence set forth inSEQ ID NO: 168, and a light chain comprising the amino acid sequence setforth in SEQ ID NO: 169.

In one embodiment, the ADC, or a pharmaceutically acceptable saltthereof, is:

wherein m is 2, Ab is either an anti-hB7-H3 antibody, wherein theanti-hB7-H3 antibody comprises a heavy chain CDR3 domain comprising theamino acid sequence set forth in SEQ ID NO: 12, a heavy chain CDR2domain comprising the amino acid sequence set forth in SEQ ID NO: 140,and a heavy chain CDR1 domain comprising the amino acid sequence setforth in SEQ ID NO: 10; and a light chain CDR3 domain comprising theamino acid sequence set forth in SEQ ID NO: 15, a light chain CDR2domain comprising the amino acid sequence set forth in SEQ ID NO: 7, anda light chain CDR1 domain comprising the amino acid sequence set forthin SEQ NO: 136; or an anti-hB7-H3 antibody, wherein the anti-hB7H3antibody comprises a heavy chain variable region comprising the aminoacid sequence set forth in SEQ ID NO: 139, and a light chain variableregion comprising the amino acid sequence set forth in SEQ ID NO: 135;or an anti-hB7-H3 antibody, wherein the anti-hB7-H3 antibody comprises aheavy chain comprising the amino acid sequence set forth in SEQ ID NO:170, and a light chain comprising the amino acid sequence set forth inSEQ ID NO: 171.

In one embodiment, the ADC, or a pharmaceutically acceptable saltthereof, is:

wherein m is 2, Ab is either an anti-hB7-H3 antibody, wherein theanti-hB7-H3 antibody comprises a heavy chain CDR3 domain comprising theamino acid sequence set forth in SEQ ID NO: 35, a heavy chain CDR2domain comprising the amino acid sequence set forth in SEQ ID NO: 34,and a heavy chain CDR1 domain comprising the amino acid sequence setforth in SEQ ID NO: 33; and a light chain CDR3 domain comprising theamino acid sequence set forth in SEQ ID NO: 39, a light chain CDR2domain comprising the amino acid sequence set forth in SEQ ID NO: 38,and a light chain CDR1 domain comprising the amino acid sequence setforth in SEQ ID NO: 37; or an anti-hB7-H3 antibody, wherein theanti-hB7H3 antibody comprises a heavy chain variable region comprisingthe amino acid sequence set forth in SEQ ID NO: 147, and a light chainvariable region comprising the amino acid sequence set forth in SEQ IDNO: 144; or an anti-hB7-11.3 antibody, wherein the anti-hB7-H3 antibodycomprises a heavy chain comprising the amino acid sequence set forth inSEQ ID NO: 168, and a light chain comprising the amino acid sequence setforth in SEQ ID NO: 169.

In one embodiment, the ADC, or a pharmaceutically acceptable saltthereof, is:

wherein m is 2, Ab is either an anti-hB7-H3 antibody, wherein theanti-hB7-H3 antibody comprises a heavy chain CDR3 domain comprising theamino acid sequence set forth in SEQ ID NO: 12, a heavy chain CDR2domain comprising the amino acid sequence set forth in SEQ ID NO: 140,and a heavy chain CDR1 domain comprising the amino acid sequence setforth in SEQ ID NO: 10; and a light chain CDR3 domain comprising theamino acid sequence set forth in SEQ ID NO: 15, a light chain CDR2domain comprising the amino acid sequence set forth in SEQ ID NO: 7, anda light chain CDR1 domain comprising the amino acid sequence set forthin SEQ ID NO: 136; or an anti-hB7-H3 antibody, wherein the anti-hB7H3antibody comprises a heavy chain variable region comprising the aminoacid sequence set forth in SEQ ID NO: 139, and a light chain variableregion comprising the amino acid sequence set forth in SEQ ID NO: 135;or an anti-hB7-H3 antibody, wherein the 7-antibody comprises a heavychain comprising the amino acid sequence set forth in SEQ ID NO: 170,and a light chain comprising the amino acid sequence set forth in SEQ IDNO: 171.

In one embodiment, the ADC, or a pharmaceutically acceptable saltthereof, is:

wherein m is 2, Ab is either an anti-hB7-H3 antibody, wherein theanti-hB7-H3 antibody comprises a heavy chain CDR3 domain comprising theamino acid sequence set forth in SEQ ID NO: 35, a heavy chain CDR2domain comprising the amino acid sequence set forth in SEQ ID NO: 34,and a heavy chain CDR1 domain comprising the amino acid sequence setforth in SEQ ID NO: 33; and a light chain CDR3 domain comprising theamino acid sequence set forth in SEQ ID NO: 39, a light chain CDR2domain comprising the amino acid sequence set forth in SEQ ID NO: 38,and a light chain CDR1 domain comprising the amino acid sequence setforth in SEQ ID NO: 37; or an anti-hB7-H3 antibody, wherein theanti-hB7H3 antibody comprises a heavy chain variable region comprisingthe amino acid sequence set forth in SEQ ID NO: 147, and a light chainvariable region comprising the amino acid sequence set forth in SEQ IDNO: 144; or an anti-hB7-H3 antibody, wherein the anti-hB7-H3 antibodycomprises a heavy chain comprising the amino acid sequence set forth inSEQ IT) NO: 168, and a light chain comprising the amino acid sequenceset forth in SEQ ID NO: 169.

In one embodiment, the ADC, or a pharmaceutically acceptable saltthereof, is:

wherein m is 2, Ab is either an anti-hB7-H3 antibody, wherein theanti-hB7-H3 antibody comprises a heavy chain CDR3 domain comprising theamino acid sequence set forth in SEQ ID NO: 12, a heavy chain CDR2domain comprising the amino acid sequence set forth in SEQ ID NO: 140,and a heavy chain CDR1 domain comprising the amino acid sequence setforth in SEQ ID NO: 10; and a light chain CDR3 domain comprising theamino acid sequence set forth in SEQ ID NO: 15, a light chain CDR2domain comprising the amino acid sequence set forth in SEQ ID NO: 7, anda light chain CDR1 domain comprising the amino acid sequence set forthin SEQ NO: 136; or an anti-hB7-H3 antibody, wherein the anti-hB7H3antibody comprises a heavy chain variable region comprising the aminoacid sequence set forth in SEQ ID NO: 139, and a light chain variableregion comprising the amino acid sequence set forth in SEQ ID NO: 135;or an anti-hB7-H3 antibody, wherein the anti-hB7-H3 antibody comprises aheavy chain comprising the amino acid sequence set forth in SEQ ID NO:170, and a light chain comprising the amino acid sequence set forth inSEQ ID NO: 171.

In one embodiment, the ADC, or a pharmaceutically acceptable saltthereof, is:

wherein m is 2, Ab is either an anti-hB7-H3 antibody, wherein theanti-hB7-H3 antibody comprises a heavy chain CDR3 domain comprising theamino acid sequence set forth in SEQ ID NO: 35, a heavy chain CDR2domain comprising the amino acid sequence set forth in SEQ ID NO: 34,and a heavy chain CDR1 domain comprising the amino acid sequence setforth in SEQ ID NO: 33; and a light chain CDR3 domain comprising theamino acid sequence set forth in SEQ ID NO: 39, a light chain CDR2domain comprising the amino acid sequence set forth in SEQ ID NO: 38,and a light chain CDR1 domain comprising the amino acid sequence setforth in SEQ ID NO: 37; or an anti-hB7-H3 antibody, wherein theanti-hB7H3 antibody comprises a heavy chain variable region comprisingthe amino acid sequence set forth in SEQ ID NO: 147, and a light chainvariable region comprising the amino acid sequence set forth in SEQ IDNO: 144; or an anti-hB7-H3 antibody, wherein the anti-hB7-H3 antibodycomprises a heavy chain comprising the amino acid sequence set forth inSEQ ID NO: 168, and a light chain comprising the amino acid sequence setforth in SEQ ID NO: 169.

In one embodiment, the ADC, or a pharmaceutically acceptable saltthereof, is:

wherein m is 2, Ab is either an anti-hB7-H3 antibody, wherein theanti-hB7-H3 antibody comprises a heavy chain CDR3 domain comprising theamino acid sequence set forth in SEQ ID NO: 12, a heavy chain CDR2domain comprising the amino acid sequence set forth in SEQ ID NO: 140,and a heavy chain CDR1 domain comprising the amino acid sequence setforth in SEQ NO: 10; and a light chain CDR3 domain comprising the aminoacid sequence set forth in SEQ ID NO: 15, a light chain CDR2 domaincomprising the amino acid sequence set forth in SEQ ID NO: 7, and alight chain CDR1 domain comprising the amino acid sequence set forth inSEQ ID NO: 136; or an anti-hB7-H3 antibody, wherein the anti-hB7H3antibody comprises a heavy chain variable region comprising the aminoacid sequence set forth in SEQ ID NO: 139, and a light chain variableregion comprising the amino acid sequence set forth in SEQ ID NO: 135;or an anti-hB7-H3 antibody, wherein the anti-hB7-H3 antibody comprises aheavy chain comprising the amino acid sequence set forth in SEQ ID NO:170, and a light chain comprising the amino acid sequence set forth inSEQ ID NO: 171.

In one embodiment, the ADC, or a pharmaceutically acceptable saltthereof, is:

wherein m is 2, Ab is either an anti-HB7-H3 antibody, wherein theanti-hB7-H3 antibody comprises a heavy chain CDR3 domain comprising theamino acid sequence set forth in SEQ ID NO: 35, a heavy chain CDR2domain comprising the amino acid sequence set forth in SEQ ID NO: 34,and a heavy chain CDR1 domain comprising the amino acid sequence setforth in SEQ ID NO: 33; and a light chain CDR3 domain comprising theamino acid sequence set forth in SEQ NO: 39, a light chain CDR2 domaincomprising the amino acid sequence set forth in SEQ ID NO: 38, and alight chain CDR1 domain comprising the amino acid sequence set forth inSEQ ID NO: 37; or an anti-hB7-H3 antibody, wherein the anti-hB7H3antibody comprises a heavy chain variable region comprising the aminoacid sequence set forth in SEQ ID NO: 147, and a light chain variableregion comprising the amino acid sequence set forth in SEQ ID NO: 144;or an anti-hB7-H3 antibody, wherein the anti-hB7-H3 antibody comprises aheavy chain comprising the amino acid sequence set forth in SEQ ID NO:168, and a light chain comprising the amino acid sequence set forth inSEQ ID NO: 169.

In one embodiment, the ADC, or a pharmaceutically acceptable saltthereof, is:

wherein m is 2, Ab is either an anti-hB7-H3 antibody, wherein theanti-hB7-H3 antibody comprises a heavy chain CDR3 domain comprising theamino acid sequence set forth in SEQ ID NO: 12, a heavy chain CDR2domain comprising the amino acid sequence set forth in SEQ ID NO: 140,and a heavy chain CDR1 domain comprising the amino acid sequence setforth in SEQ ID NO: 10; and a light chain CDR3 domain comprising theamino acid sequence set forth in SEQ ID NO: 15, a light chain CDR2domain comprising the amino acid sequence set forth in SEQ NO: 7, and alight chain CDR1 domain comprising the amino acid sequence set forth inSEQ ID NO: 136; or an anti-hB7-H3 antibody, wherein the anti-hB7H3antibody comprises a heavy chain variable region comprising the aminoacid sequence set forth in SEQ ID NO: 139, and a light chain variableregion comprising the amino acid sequence set forth in SEQ ID NO: 135;or an anti-hB7-H3 antibody, wherein the anti-hB7-H3 antibody comprises aheavy chain comprising the amino acid sequence set forth in SEQ ID NO:170, and a light chain comprising the amino acid sequence set forth inSEQ ID NO: 171.

Bcl-xL inhibitors, including warheads and synthons, and methods ofmaking the same, are described in US 2016/0339117 (AbbVie Inc.), whichis incorporated by reference herein.

III.A.4. Methods of Synthesis of Bcl-xL ADCs

The Bcl-xL inhibitors and synthons described herein may be synthesizedusing standard, known techniques of organic chemistry. General schemesfor synthesizing Bcl-xL inhibitors and synthons that may be used as-isor modified to synthesize the full scope of Bcl-xL inhibitors andsynthons described herein are provided below. Specific methods forsynthesizing exemplary Bcl-xL inhibitors and synthons that may be usefulfor guidance are provided in the Examples section. ADCs may likewise beprepared by standard methods, such as methods analogous to thosedescribed in Hamblett et al., 2004, “Effects of Drug Loading on theAntitumor Activity of a Monoclonal Antibody Drug Conjugate”, Clin.Cancer Res. 10:7063-7070; Doronina et al., 2003, “Development of potentand highly efficacious monoclonal antibody auristatin conjugates forcancer therapy,” Nat. Biotechnol. 21(7):778-784; and Francisco et al.,2003, Blood 102:1458-1465. For example, ADCs with four drugs perantibody may be prepared by partial reduction of the antibody with anexcess of a reducing reagent such as DTT or TCEP at 37° C. for 30 min,then the buffer exchanged by elution through SEPHADEX® G-25 resin with 1mM DTPA in DPBS. The eluent is diluted with further DPBS, and the thiolconcentration of the antibody may be measured using5,5′-dithiobis(2-nitrobenzoic acid) [Ellman's reagent]. An excess, forexample 5-fold, of a linker-drug synthon is added at 4° C. for 1 hr, andthe conjugation reaction may be quenched by addition of a substantialexcess, for example 20-fold, of cysteine. The resulting ADC mixture maybe purified on SEPHADEX G-25 equilibrated in PBS to remove unreactedsynthons, desalted if desired, and purified by size-exclusionchromatography. The resulting ADC may then be then sterile filtered, forexample, through a 0.2 μm filter, and lyophilized if desired forstorage. In certain embodiments, all of the interchain cysteinedisulfide bonds are replaced by linker-drug conjugates. One embodimentpertains to a method of making an ADC, comprising contacting a synthondescribed herein with an antibody under conditions in which the synthoncovalently links to the antibody Examples of the foregoing Bcl-xLinhibitors, linkers, and synthons thereof, as well as methods of makingthe same, can be found in US Patent Publication No. US 2016/0339117, theentire contents of which are incorporated by reference herein.

Specific methods for synthesizing exemplary ADCs that may be used tosynthesize the full range of ADCs described herein are provided in theExamples section.

III.A.5. General Methods for Synthesizing Bcl-xL Inhibitors

In the schemes below, the various substituents Ar¹, Ar², Z¹, R⁴, R¹⁰,R^(11a) and R^(11b) are as defined in the Detailed Description section.

5.1.1. Synthesis of Compound (6)

The synthesis of an intermediate (6) is described in Scheme 1. Compound(1) can be treated with BH₃.THF to provide compound (2). The reaction istypically performed at ambient temperature in a solvent, such as, butnot limited to, tetrahydrofuran. Compound (3) can be prepared bytreating compound (2) with

in the presence of cyanomethylenetributylphosphorane. The reaction istypically performed at an elevated temperature in a solvent such as, butnot limited to, toluene. Compound (3) can be treated withethane-1,2-diol in the presence of a base such as, but not limited to,triethylamine, to provide compound (4). The reaction is typicallyperformed at an elevated temperature, and the reaction may be performedunder microwave conditions. Compound (4) can be treated with a strongbase, such as, but not limited to, n-butyllithium, followed by theaddition of iodomethane, to provide compound (5). The addition andreaction is typically performed in a solvent such as, but not limitedto, tetrahydrofuran, at a reduced temperature before warming up toambient temperature for work up. Compound (5) can be treated withN-iodosuccinimide to provide compound (6). The reaction is typicallyperformed at ambient temperature is a solvent such as, but not limited,to, N,N-dimethylformamide.

5.1.2. Synthesis of Compound (12)

The synthesis of intermediate (12) is described in Scheme 2. Compound(3) can be treated with tri-n-butyl-allylstannane in the presence ofZnCl₂.Et₂O or N, N′-azoisobutyronitrile (AIBN) to provide compound (10)(Yamamoto et al., 1998, Heterocycles 47:765-780). The reaction istypically performed at −78° C. in a solvent, such as, but not limited todichloromethane. Compound (10) can be treated under standard conditionsknown in the art for hydroboration/oxidation to provide compound (11).For example, treatment of compound (10) with a reagent such as BH₃.THFin a solvent such as, but not limited to, tetrahydrofuran followed bytreatment of the intermediate alkylborane adduct with an oxidant suchas, but not limited to, hydrogen peroxide in the presence of a base suchas, but not limited to, sodium hydroxide would provide compound (11)(Brown et al., 1968, J. Am. Chem. Soc. 86:397). Typically the additionof BH₃.THF is performed at low temperature before, warming to ambienttemperature, which is followed by the addition of hydrogen peroxide andsodium hydroxide to generate the alcohol product. Compound (12) can begenerated according to Scheme 1, as previously described for compound(6).

5.1.3. Synthesis of Compound (15)

The synthesis of intermediate (15) is described in Scheme 3, Compound(3) can be reacted with thiourea in a solvent mixture of acetic acid and48% aqueous HBr solution at 100° C. to yield an intermediate that can besubsequently treated with sodium hydroxide in a solvent mixture such as,but not limited to, 20% v/v ethanol in water to provide compound (13).Compound (13) can be reacted with 2-chloroethanol in the presence of abase such as, but not limited to, sodium ethoxide to provide compound(14). The reaction is typically performed at ambient or elevatedtemperatures in a solvent such as, but not limited to, ethanol. Compound(15) can be generated according to Scheme 1, as previously described forcompound (6).

5.1.4. Synthesis of Compound (22)

The synthesis of compound (22) is described in Scheme 4. Compound (16)can be reacted with iodomethane in the presence of a base such as, butnot limited to, potassium carbonate to provide compound (17). Thereaction is typically conducted at ambient or elevated temperature in asolvent such as, but not limited to, acetone or N,N-dimethylformamide.Compound (17) can be reacted under photochemical conditions with tosylcyanide in the presence of benzophenone to provide compound (18) (seeKamijo et al., 2011, Org. Lett., 13:5928-5931). The reaction istypically run at ambient temperature in a solvent such as, but notlimited to, acetonitrile or benzene using a Riko 100 W medium pressuremercury lamp as the light source. Compound (18) can be reacted withlithium hydroxide in a solvent system such as, but not limited to,mixtures of water and tetrahydrofuran or water and methanol to providecompound (19). Compound (19) can be treated with BH₃.THF to providecompound (20). The reaction is typically performed at ambienttemperature in a solvent, such as, but not limited to, tetrahydrofuran.Compound (21) can be prepared by treating compound (20) with

in the presence of cyanomethylenetributylphosphorane. The reaction istypically performed at an elevated temperature in a solvent such as, butnot limited to, toluene. Compound (21) can be treated withN-iodosuccinimide to provide compound (22). The reaction is typicallyperformed at ambient temperature is a solvent such as, but not limitedto, N,N-dimethylformamide.

5.1.5. Synthesis of Compound (24)

The synthesis of pyrazole compound (24), is described in Scheme 5.Compound (22) can be treated with a reducing agent such as, but notlimited to, lithium aluminum hydride in a solvent such as, but notlimited to, diethyl, ether or tetrahydrofuran to provide compound (23).Typically the reaction is performed at 0° C. before warming to ambientor elevated temperature. Compound (23) can be reacted with di-tert-butyldicarbonate under standard conditions described herein or in theliterature to provide compound (24).

5.1.6. Synthesis of Compound (24a)

The synthesis of intermediate (24a) is described in Scheme 6. Compound(22a) can be hydrolyzed using conditions described in the literature toprovide compound (23a). Typically the reaction is run in the presence ofpotassium hydroxide in a solvent such as, but not limited to, ethyleneglycol at elevated temperatures (see Roberts et al., 1994, J. Org. Chem.59:6464-6469; Yang et al, 2013, Org. Lett, 15:690-693). Compound (24a)can be made from compound (23a) by Curtius rearrangement usingconditions described in the literature. For example, compound (23a) canbe reacted with sodium azide in the presence of tetrabutylammoniumbromide, zinc(II) triflate and di-ten-butyl dicarbonate to providecompound (24a) (see Lebel et al., Org. Lett, 2005, 7:4107-4110),Typically the reaction is run at elevated temperatures, preferably from40-50° C., in a solvent such as, but not limited to, tetrahydrofuran.

5.1.7. Synthesis of Compound (29)

As shown in Scheme 7, compounds of formula (27) can be prepared byreacting compounds of formula (25) with tort-butyl3-bromo-6-fluoropicolinate (26) in the presence of a base, such as, butnot limited to, N,N-diisopropylethylamine, or triethylamine. Thereaction is typically performed under an inert atmosphere at an elevatedtemperature in a solvent, such as, but not limited to, dimethylsulfoxide. Compounds of formula (27) can be reacted with4,4,5,5-tetramethyl-1,3,2-dioxaborolane (28), under borylationconditions described herein or in the literature to provide compounds offormula (29).

5.1.8. Synthesis of Compound (38)

Scheme 8 describes a method to make intermediates which contain -Nu(nucleophile) tethered to an adamantane and picolinate protected as at-butyl ester. Compound (30) can be reacted with compound (31) underSuzuki Coupling conditions described herein or in the literature toprovide methyl compound (32). Compound (32) can be treated with a basesuch as but not limited to triethylamine, followed by methanesulfonylchloride to provide compound (33). The addition is typically performedat low temperature before warming up to ambient temperature in asolvent, such as, but not limited to, dichloromethane. Compound (33) canbe reacted with a nucleophile (Nu) of formula (34) to provide compound(35). Examples of nucleophiles include, but are not limited to, sodiumazide, methylamine, ammonia and di-tert-butyl iminodicarbonate. Compound(17) can be reacted with lithium hydroxide to provide compound (36). Thereaction is typically performed at ambient temperature in a solvent suchas but not limited to tetrahydrofuran, methanol, water, or mixturesthereof. Compound (36) can be reacted with compound (37) under amidationconditions described herein or readily available in the literature toprovide compounds of formula (38).

5.1.9. Synthesis of Compounds (42) and (43)

Scheme 9 shows representative methods used to make solubilized Bcl-xLinhibitors. Bcl-XL inhibitors can be synthesized using the generalapproach of modifying a primary amine with a solubilizing group and thenattaching the resulting secondary amine to a linker as described inlater schemes. For example, compound (41) can be prepared by reactingcompound (39) with compound (40). The reaction is typically performed atambient temperature in a solvent such as but not limited toN,N-dimethylformamide. Compound (41) can be reacted with trifluoroaceticacid to provide compound (43). The reaction is typically performed atambient temperature in a solvent such as but not limited todichloromethane. Another example shown in Scheme 9 is the reaction ofcompound (39) with diethyl vinylphosphonate, followed by reaction withbromotrimethylsilane and allyltrimethylsilane to provide compound (42).Other examples to introduce solubilizing groups on the Bcl-xL inhibitorsdescribed herein include, but are not limited to, reductive aminationreactions, alkylations, and amidation reactions.

5.1.10. Synthesis of Compound (47)

Scheme 10 shows introduction of a solubilizing group by amidationreaction. Bcl-xL inhibitors can be synthesized using the generalapproach of modifying a primary or secondary amine with a solubilizinggroup and then attaching the resulting amine to a linker as described inlater schemes. For example, compound (45) can be treated sequentiallywith HATU and compound (44), to provide compound (46). Compound (46) canbe treated with diethylamine in solvents such as, but not limited to,N,N-dimethylformamide to give compound (47).

5.1.11. Synthesis of Compound (51)

Scheme 11 shows representative methods to make solubilized Bcl-xLinhibitors. Bcl-xL inhibitors can be synthesized using the generalapproach of modifying a primary amine with a spacer to give adifferentially protected diamine. The unprotected secondary amine can bemodified with a solubilizing group. Deprotection of a protected aminethem reveals a site for linker attachment, as described in laterschemes. For example, compound (39) can be reductively alkylated withreagents such as, but not limited to tert-butyl4-oxopiperidine-1-carboxylate (48), under conditions known in the art,to provide a secondary amine (49). Compound (50) can be prepared byreacting compound (49) with4-((tert-butyidiphenylsilyl)oxy)-2,2-dimethylbutyl ethanesulfonate (40).The reaction is typically performed at ambient temperature in a solventsuch as but not limited to N,N-dimethylformamide. Compound (40) can bereacted with trifluoroacetic acid to provide compound (Si). The reactionis typically performed at ambient temperature in a solvent such as butnot limited to dichloromethane.

5.1.12. Synthesis of Compound (61)

Scheme 12 describes a method to synthesize solubilized Bcl-xLinhibitors, Compound (52) can be reacted with methanesulfonyl chloride,in the presence of a base, such as, but not limited to, triethylamine,to provide compound (53). The reaction is typically performed at a lowtemperature in a solvent such as but not limited to dichloromethane.Compound (53) can be treated with ammonia in methanol to providecompound (54). The reaction is typically performed at an elevatedtemperature, and the reaction may be performed under microwaveconditions. Compound (56) can be prepared by reacting compound (55) inthe presence of a base such as but not limited toN,N-diisopropylethylamine. The reaction is typically performed atambient temperature in a solvent such as but not limited toN,N-dimethylformamide. Compound (56) can be treated withdi-t-butyldicarbonate and 4-(dimethylamino)pyridine to provide compound(57). The reaction is typically performed at ambient temperature in asolvent such as but not limited to tetrahydrofuran. Compound (59) can beprepared by reacting compound (57) with a boronate ester (or theequivalent boronic acid) of formula (58), under Suzuki Couplingconditions described herein or in the literature.Bis(2,5-dioxopyrrolidin-1-yl) carbonate can be reacted with compound(37), followed by reaction with compound (59), to provide compound (60).The reaction is typically performed at ambient temperature in a solventsuch as, but not limited to, acetonitrile. Compound (61) can be preparedby treating compound (60) with trifluoroacetic acid. The reaction istypically performed at ambient temperature in a solvent such as but notlimited to dichloromethane.

5.1.13. Synthesis of Compound (70)

Scheme 13 describes the synthesis of 5-hydroxy tetrahydroisoquinolineintermediates, Compound (63) can be prepared by treating compound (62)with N-bromosuccinimide. The reaction is typically performed at ambienttemperature is a solvent such as, but not limited to,N,N-dimethylformamide. Compound (63) can be reacted with benzyl bromidein the presence of a base, such as, but not limited to, potassiumcarbonate, to provide compound (64). The reaction is typically performedat an elevated temperature, in a solvent such as, but not limited to,acetone. Compound (64) can be treated with carbon monoxide and methanolin the presence of a base, such as, but not limited to, triethylamine,and a catalyst, such as, but not limited to, compound (65). The reactionis typically performed at an elevated temperature under an inertatmosphere. Compound (65) can be treated with an acid, such as, but notlimited to, hydrochloric acid in dioxane, to provide compound (66). Thereaction is typically performed at ambient temperature in a solvent,such as, but not limited to, tetrahydrofuran. Compound (67) can beprepared by reacting compound (66) with tert-butyl3-bromo-6-fluoropicolinate in the presence of a base, such as, but notlimited to, triethylamine. The reaction is typically performed under aninert atmosphere at an elevated temperature in a solvent, such as, butnot limited to, dimethyl sulfoxide. Compound (67) can be reacted with aboronic acid of formula (68), wherein Ad is the methyladamantane moietyof the compounds of the disclosure (e.g., the compounds of formulae(Ha)-(Hd)), under Suzuki Coupling conditions described herein or in theliterature to provide compound (69). Compound (70) can be prepared byreacting compound (69) with hydrogen in the presence of Pd(OH)₂. Thereaction is typically performed at an elevated temperature in a solventsuch as, but not limited to tetrahydrofuran.

5.1.14. Synthesis of Compound (75)

Scheme 14 shows representative methods used to make solubilized Bcl-xLinhibitors. Bcl-xL inhibitors can be synthesized using the generalapproach of modifying an Ar² substituent with a solubilizing group andthen attaching an amine to a linker as described in later schemes. Forexample, compound (71) can be reacted with tea-butyl 2-bromoacetate inthe presence of a base such as, but not limited to, potassium carbonatein a solvent such as, but not limited, to N,N-dimethylformamide.Compound (72) can be treated with aqueous lithium hydroxide in a solventsuch as, but not limited to, methanol, tetrahydrofuran or mixturesthereof to provide compound (73). Compound (74) can be obtained byamidation of compound (73) with compound (37) under conditionspreviously described. Compound (74) can be treated with acids such as,but not limited to trifluoroacetic acid or HCl, to provide a Bcl-xLinhibitor of the formula (75). The reaction is typically performed atambient temperature in solvents such as, but not limited to,dichloromethane or 1,4-dioxane.

III.A.6. General Methods for Synthesizing Bcl-xL Synthons

In the schemes below, the various substituents Ar¹, Ar², Z¹, Y, G,R^(11a) and R^(11b) are as defined in the Detailed Description section.

5.2.1. Synthesis of Compound (89)

As shown in scheme 15, compounds of formula (77), wherein PG is anappropriate base labile protecting group and AA(2) is Cit, Ala, or Lys,can be reacted with 4-(aminophenyl)methanol (78), under amidationconditions described herein or readily available in the literature toprovide compound (79). Compound (80) can be prepared by reactingcompound (79) with a base such as, but not limited to, diethylamine. Thereaction is typically performed at ambient temperature in a solvent suchas but not limited to N,N-dimethylformamide. Compound (81), wherein PGis an appropriate base or acid labile protecting group and AA(1) is Valor Phe, can be reacted with compound (80), under amidation conditionsdescribed herein or readily available in the literature to providecompound (82). Compound (83) can be prepared by treating compound (82)with diethylamine or trifluoroacetic acid, as appropriate. The reactionis typically performed at ambient temperature in a solvent such as butnot limited to dichloromethane. Compound (84), wherein Sp is a spacer,can be reacted with compound (83) to provide compound (85). The reactionis typically performed at ambient temperature in a solvent such as butnot limited to N,N-dimethylformamide. Compound (85) can be reacted withbis(4-nitrophenyl) carbonate (86) in the presence of a base such as, butnot limited to N,N-diisopropylethylamine to provide compounds (87). Thereaction is typically performed at ambient temperature in a solvent suchas but not limited to N,N-dimethylformamide. Compounds (87) can bereacted with compound (88) in the presence of a base such as, but notlimited to, N,N-diisopropylethylamine, to provide compound (89). Thereaction is typically performed at ambient temperature in a solvent suchas, but not limited to, N,N-dimethylformamide.

5.2.2. Synthesis of Compounds (94) and (96)

Scheme 16 describes the installment of alternative mAb-linkerattachments to dipeptide Synthons. Compound (88) can be reacted withcompound (90) in the presence of a base such as, but not limited to,N,N-diisopropylamine to provide compound (91). The reaction is typicallyperformed at ambient temperature in a solvent such as but not limited toN,N-dimethylformamide. Compound (92) can be prepared by reactingcompound (91) with diethylamine. The reaction is typically performed atambient temperature in a solvent such as but not limited toN,N-dimethylformamide. Compound (93), wherein X¹ is Cl, Br, or I, can bereacted with compound (92), under amidation conditions described hereinor readily available in the literature to provide compound (94).Compound (92) can be reacted with compounds of formula (95) underamidation conditions described herein or readily available in theliterature to provide compound (96).

5.2.3. Synthesis of Compound (106)

Scheme 17 describes the synthesis of vinyl glucuronide linkerintermediates and synthons.(2R,3R,4S,5S,6S)-2-Bromo-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (97) can be treated with silver oxide, followed by4-bromo-2-nitrophenol (98) to provide(2S,3R,4S,5S,6S)-2-(4-bromo-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (99). The reaction is typically performed at ambienttemperature in a solvent, such as, but not limited to, acetonitrile.(2S,3R,4S,5S,6S)-2-(4-Bromo-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (99) can be reacted with(E)-tert-butyldimethyl((3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)ally)oxy)silane(100) in the presence of a base such as, but not limited to, sodiumcarbonate, and a catalyst such as but not limited totris(dibenzylideneacetone)dipalladium (Pd₂(dba)₃), to provide(2S,3R,4S,5S,6S)-2-(4-((E)-3-((tert-butyldimethylsilyl)oxy)prop-1-en-1-yl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (101). The reaction is typically performed at an elevatedtemperature in a solvent, such as, but not limited to, tetrahydrofuran.(2S,3R,4S,5S,6S)-2-(2-amino-4-((E)-3-hydroxyprop-1-en-1-yl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (102) can be prepared by reacting(25,3R,4S,5S,6S)-2-(4-((E)-3-((tert-butyldimethylsilyl)oxy)prop-1-en-1-yl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (101) with zinc in the presence of an acid such as, but notlimited to, hydrochloric acid. The addition is typically performed atlow temperature before warming to ambient temperature in a solvent suchas, but not limited to, tetrahydrofuran, water, or mixtures thereof.(2S,3R,4S,5S,6S)-2-(2-amino-4-((E)-3-hydroxyprop-1-en-1-yl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (102) can be reacted with (9H-fluoren-9-yl)methyl(3-chloro-3-oxopropyl)carbamate (103), in the presence of a base suchas, but not limited to, N,N-diisopropylethylamine, to provide(2S,3R,4S,5S,6S)-2-(2-(3-(4(9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-((E)-3-hydroxyprop-1-en-1-yl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (104). The addition is typically performed at low temperaturebefore warming to ambient temperature in a solvent such as, but notlimited to, dichloromethane. Compound (88) can be reacted with(2S,3R,4S,5S,6S)-2-(2-(34(49H-fluoren-9-yl)methoxycarbonyl)amino)propanamido)-4-((E)-3-hydroxyprop-1-en-1-yl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (104) in the presence of a base such as, but not limited to,N-ethyl-N-isopropylpropan-2-amine, followed by work up and reaction withcompound (105) in the presence of a base such as, but not limited to,N,N-diisopropylethylamine to provide compound (106). The reactions aretypically performed at ambient temperature in a solvent such as, but notlimited to N,N-dimethylformamide.

5.2.4. Synthesis of Compound (115)

Scheme 18 describes the synthesis of a representative 2-etherglucuronide linker intermediate and synthon.(2S,3R,4S,5S,6S)-2-Bromo-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (97) can be reacted with 2,4-dihydroxybenzaldehyde (107) inthe presence of silver carbonate to provide(2S,3R,4S,5S,6S)-2-(4-formyl-3-hydroxyphenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (108). The reaction is typically performed at an elevatedtemperature in a solvent, such as, but not limited to, acetonitrile.(2S,3R,4S,5S,6S)-2-(4-Formyl-3-hydroxyphenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (108) can be treated with sodium borohydride to provide(2S,3R,4S,5S,6S)-2-(3-hydroxy-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (109). The addition is typically performed at low temperaturebefore warming to ambient temperature in a solvent such as but notlimited to tetrahydrofuran, methanol, or mixtures thereof.(2S,3R,4S,5S,6S)-2-(4-(((tert-butyldimethylsilyl)oxy)methyl)-3-hydroxyphenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triltriacetate (110) can be prepared by reacting(25,3R,4S,5S,6S)-2-(3-hydroxy-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (109) with tert-butyldimethylsilyl chloride in the presenceof imidazole. The reaction is typically performed at low temperature ina solvent, such as, but not limited to, dichloromethane.(2S,3R,4S,5S,6S)-2-(3-(2-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)ethoxy)ethoxy)-4-(((tert-butyldimethylsilyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (111) can be prepared by reacting(2S,3R,4S,5S,6S)-2-(4-(((tert-butyldimethylsilyl)oxy)methyl)-3-hydroxyphenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (110) with (9H-fluoren-9-yl)methyl(2-(2-hydroxyethoxy)ethyl)carbamate in the presence oftriphenylphosphine and a azodicarboxylate such as, but not limited to,di-tert-butyl diazene-1,2-dicarboxylate. The reaction is typicallyperformed at ambient temperature in a solvent such as but not limited totoluene.(2S,3R,4S,5S,6S)-2-(3-(2-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)ethoxy)ethoxy)-4-(((tert-butyldimethylsilyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (111) can be treated with acetic acid to provide(2S,3R,4S,5S,6S)-2-(3-(2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethoxy)ethoxy)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (112). The reaction is typically performed at ambienttemperature in a solvent such as but not limited to water,tetrahydrofuran, or mixtures thereof.(2S,3R,4S,5S,6S)-2-(3-(2-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)ethoxy)ethoxy)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (113) can be prepared by reacting(2S,3R,4S,5S,6S)-2-(3-(2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethoxy)ethoxy)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (112) with bis(4-nitrophenyl) carbonate in the presence of abase such as but not limited to N-ethyl-N-isopropylpropan-2-amine. Thereaction is typically performed at ambient temperature in a solvent suchas but not limited to N,N-dimethylformamide.(2S,3R,4S,5S,6S)-2-(3-(2-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)ethoxy)ethoxy)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (113) can be treated with compound (88) in the presence of abase such as but not limited to N-ethyl-N-isopropylpropan-2-amine,followed by treatment with lithium hydroxide to provide a compound(114). The reaction is typically performed at ambient temperature in asolvent such as but not limited to N,N-dimethylformamide,tetrahydrofuran, methanol, or mixtures thereof. Compound (115) can beprepared by reacting compound (114) with compound (84) in the presenceof a base such as but not limited to N-ethyl-N-isopropylpropan-2-amine.The reaction is typically performed at ambient temperature in a solventsuch as but not limited to N,N-dimethylformamide.

5.2.5. Synthesis of Compound (119)

Scheme 19 describes the introduction of a second solubilizing group to asugar linker. Compound (116) can be reacted with(R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-sulfopropanoic acid(117), under amidation conditions described herein or readily availablein the literature, followed by treatment with a base such as but notlimited to diethylamine, to provide compound (118). Compound (118) canbe reacted with compound (84), wherein Sp is a spacer, under amidationconditions described herein or readily available in the literature, toprovide compound (119).

5.2.6. Synthesis of Compound (129)

Scheme 20 describes the synthesis of 4-ether glucuronide linkerintermediates and synthons.4-(2-(2-Bromoethoxy)ethoxy)-2-hydroxybenzaldehyde (122) can be preparedby reacting 2,4-dihydroxybenzaldehyde (120) with1-bromo-2-(2-bromoethoxy)ethane (121) in the presence of a base such as,but not limited to, potassium carbonate. The reaction is typicallyperformed at an elevated temperature in a solvent such as but notlimited to acetonitrile.4-(2-(2-Bromoethoxy)ethoxy)-2-hydroxybenzaldehyde (122) can be treatedwith sodium azide to provide4-(2-(2-azidoethoxy)ethoxy)-2-hydroxybenzaldehyde (123). The reaction istypically performed at ambient temperature in a solvent such as but notlimited to N,N-dimethylformamide.(2S,3R,4S,5S,6S)-2-(5-(2-(2-Azidoethoxy)ethoxy)-2-formylphenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (125) can be prepared by reacting4-(2-(2-azidoethoxy)ethoxy)-2-hydroxybenzaldehyde (123) with(3R,4S,5S,6S)-2-bromo-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (124) in the presence of silver oxide. The reaction istypically performed at ambient temperature in a solvent such as, but notlimited to, acetonitrile. Hydrogenation of(2S,3R,4S,5S,6S)-2-(5-(2-(2-azidoethoxy)ethoxy)-2-formylphenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (125) in the presence of Pd/C will provide(2S,3R,4S,5S,6S)-2-(5-(2-(2-amino)ethoxy)ethoxy)-2-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (126). The reaction is typically performed at ambienttemperature in a solvent such as, but not limited to, tetrahydrofuran.(2S,3R,4S,5S,6S)-2-(5-(2-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)ethoxy)ethoxy)-2-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (127) can be prepared by treating(2S,3R,4S,5S,6S)-2-(5-(2-(2-amino)ethoxy)ethoxy)-2-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (126) with (9H-fluoren-9-yl)methyl carbonochloridate in thepresence of a base, such as, but not limited to,N-ethyl-N-isopropylpropan-2-amine. The reaction is typically performedat low temperature in a solvent such as, but not limited to,dichloromethane. Compound (88) can be reacted with(2S,3R,4S,5S,6S)-2-(5-(2-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)ethoxy)ethoxy)-2-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (127) in the presence of a base, such as, but not limited to,N-ethyl-N-isopropylpropan-2-amine, followed by treatment with lithiumhydroxide to provide compound (128). The reaction is typically performedat low temperature in a solvent such as, but not limited to,N,N-dimethylformamide. Compound (129) can be prepared by reactingcompound (128) with compound (84) in the presence of a base such as, butnot limited to, N-ethyl-N-isopropylpropan-2-amine. The reaction istypically performed at ambient temperature in a solvent such as but notlimited to N,N-dimethylformamide.

5.2.7. Synthesis of Compound (139)

Scheme 21 describes the synthesis of carbamate glucuronide intermediatesand synthons. 2-Amino-5-(hydroxymethyl)phenol (130) can be treated withsodium hydride and then reacted with 2-(2-azidoethoxy)ethyl4-methylbenzenesulfonate (131) to provide(4-amino-3-(2-(2-azidoethoxy)ethoxy)phenyl)methanol (132). The reactionis typically performed at an elevated temperature in a solvent such as,but not limited to N,N-dimethylformamide.2-(2-(2-Azidoethoxy)ethoxy)-4-(((tert-butyldimethylsilyl)oxy)methyl)aniline(133) can be prepared by reacting(4-amino-3-(2-(2-azidoethoxy)ethoxy)phenyl)methanol (132) withtert-butyldimethylchlorosilane in the presence of imidazole. Thereaction is typically performed at ambient temperature in a solvent suchas, but not limited to tetrahydrofuran.2-(2-(2-Azidoethoxy)ethoxy)-4-(((tert-butyldimethylsilyl)oxy)methyl)aniline(133) can be treated with phosgene, in the presence of a base such asbut not limited to triethylamine, followed by reaction with(3R,4S,5S,6S)-2-hydroxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (134) in the presence of a base such as but not limited totriethylamine, to provide2S,3R,4S,5S,6S)-2-(((2-(2-(2-azidoethoxy)ethoxy)-4-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)carbamoyl)oxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (135). The reaction is typically performed in a solvent suchas, but not limited to, toluene, and the additions are typicallyperformed at low temperature, before warming up to ambient temperatureafter the phosgene addition and heating at an elevated temperature afterthe(3R,4S,5S,6S)-2-hydroxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (134) addition.(2S,3R,4S,5S,6S)-2-(((2-(2-(2-Azidoethoxy)ethoxy)-4-(hydroxymethyl)phenyl)carbamoyl)oxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (136) can be prepared by reacting2S,3R,4S,5S,6S)-2-(((2-(2-(2-azidoethoxy)ethoxy)-4-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)carbamoyl)oxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (135) with p-toluenesulfonic acid monohydrate. The reactionis typically performed at ambient temperature in a solvent such as, butnot limited to methanol.(2S,3R,4S,5S,6S)-2-(((2-(2-(2-Azidoethoxy)ethoxy)-4-(hydroxymethyl)phenyl)carbamoyl)oxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (136) can be reacted with bis(4-nitrophenyl)carbonate in thepresence of a base such as, but not limited to,N,N-diisopropylethylamine, to provide(2S,3R,4S,5S,6S)-2-(((2-(2-(2-azidoethoxy)ethoxy)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)carbamoyl)oxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (137). The reaction is typically performed at ambienttemperature in a solvent such as, but not limited to,N,N-dimethylformamide.(2S,3R,4S,5S,6S)-2-(((2-(2-(2-Azidoethoxy)ethoxy)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)carbamoyl)oxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (137) can be reacted with compound in the presence of a basesuch as, but not limited to, N,N-diisopropylethylamine, followed bytreatment with aqueous lithium hydroxide, to provide compound (138). Thefirst step is typically conducted at ambient temperature in a solventsuch as, but not limited to N,N-dimethylformamide, and the second stepis typically conducted at low temperature in a solvent such as but notlimited to methanol.

Compound (138) can be treated with tris(2-carboxyethyl))phosphinehydrochloride, followed by reaction with compound (84) in the presenceof a base such as, but not limited to, N,N-diisopropylethylamine, toprovide compound (139). The reaction with tris(2-carboxyethyl))phosphinehydrochloride is typically performed at ambient temperature in a solventsuch as, but not limited to, tetrahydrofuran, water, or mixturesthereof, and the reaction with N-succinimidyl 6-maleimidohexanoate istypically performed at ambient temperature in a solvent such as, but notlimited to, N,N-dimethylformamide.

5.2.8. Synthesis of Compound (149)

Scheme 22 describes the synthesis of galactoside linker intermediatesand synthons.(2S,3R,4S,5S,6R)-6-(Acetoxymethyl)tetrahydro-2H-pyran-2,3,4,5-tetrayltetraacetate (140) can be treated with HBr in acetic acid to provide(2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-bromotetrahydro-2H-pyran-3,4,5-triyltriacetate (141). The reaction is typically performed at ambienttemperature under a nitrogen atmosphere.(2R,3S,4S,5R,6S)-2-(Acetoxymethyl)-6-(4-formyl-2-nitrophenoxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate (143) can be prepared by treating(2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-bromotetrahydro-2H-pyran-3,4,5-triyltriacetate (141) with silver( ) oxide in the presence of4-hydroxy-3-nitrobenzaldehyde (142). The reaction is typically performedat ambient temperature in a solvent such as, but not limited to,acetonitrile.(2R,3S,4S,5R,6S)-2-(Acetoxymethyl)-6-(4-formyl-2-nitrophenoxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate (143) can be treated with sodium borohydride to provide(2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(4-(hydroxymethyl)-2-nitrophenoxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate (144). The reaction is typically performed at low temperaturein a solvent such as but not limited to tetrahydrofuran, methanol, ormixtures thereof.(2R,3S,4S,5R,6S)-2-(Acetoxymethyl)-6-(2-amino-4-(hydroxymethyl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate (145) can be prepared by treating(2R,3S,4S,5R,6S)-2-(acetoxymethyl)-(4-(hydroxymethyl)-2-nitrophenoxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate (144) with zinc in the presence of hydrochloric acid. Thereaction is typically performed at low temperature, under a nitrogenatmosphere, in a solvent such as, but not limited to, tetrahydrofuran.(2S,3R,4S,5S,6R)-2-(2-(3-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-(hydroxymethyl)phenoxy)-6-(acetoxymethyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (146) can be prepared by reacting(2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(2-amino-4-(hydroxymethyl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate (145) with (9H-fluoren-9-yl)methyl(3-chloro-3-oxopropyl)carbamate (103) in the presence of a base such as,but not limited to, N,N-diisopropylethylamine. The reaction is typicallyperformed at low temperature, in a solvent such as, but not limited to,dichloromethane.(2S,3R,4S,5S,6R)-2-(2-(3-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-(hydroxymethyl)phenoxy)-6-(acetoxymethyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (146) can be reacted with bis(4-nitrophenyl)carbonate in thepresence of a base such as, but not limited to,N,N-diisopropylethylamine, to provide(2S,3R,4S,5S,6R)-2-(2-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(acetoxymethyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (147). The reaction is typically performed at lowtemperature, in a solvent such as, but not limited to,N,N-dimethylformamide.(2S,3R,4S,5S,6R)-2-(2-(3-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(acetoxymethyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (147) can be reacted with compound (88) in the presence of abase such as, but not limited to N,N-diisopropylethylamine, followed bytreatment with lithium hydroxide, to provide compound (148). The firststep is typically performed at low temperature, in a solvent such as,but not limited to, N,N-dimethylformamide, and the second step istypically performed at ambient temperature, in a solvent such as, butnot limited to, methanol. Compound (148) can be treated with compound(84), wherein Sp is a spacer, in the presence of a base, such as, butnot limited to N,N-diisopropylethylamine, to provide compound (149). Thereaction is typically performed at ambient temperature, in a solventsuch as, but not limited to, N,N-dimethylformamide.

IIA.7. General Methods for Synthesizing Anti-B7-H3 ADCs

The present invention also discloses a process to prepare an anti-B7-H3ADC according to structural formula (I):

(D-L-LK_(m)Ab  (I)

wherein D, L, LK, Ab and m are as defined in the Detailed Descriptionsection. The process comprises:

treating an antibody in an aqueous solution with an effective amount ofa disulfide reducing agent at 30-40° C. for at least 15 minutes, andthen cooling the antibody solution to 20-27° C.;

adding to the reduced antibody solution a solution of water/dimethylsulfoxide comprising a synthon selected from the group of 2.1 to 2.176(Table B):

adjusting the pH of the solution to a pH of 7.5 to 8.5:

allowing the reaction to run for 48 to 80 hours to form the ADC;

wherein the mass is shifted by 18±2 amu for each hydrolysis of asuccinimide to a succinamide as measured by electron spray massspectrometry; and

wherein the ADC is optionally purified by hydrophobic interactionchromatography.

In certain embodiments, the antibody is an hB7-H3 antibody, wherein thehB7-H3 antibody comprises the heavy and light chain CDRs of huAb3v2.5,huAb3v2.6, or huAb13v1.

The present invention is also directed to an anti-B7-H3 ADC prepared bythe above-described process.

In certain embodiments, the anti-B7-H3 ADC disclosed in the presentapplication is formed by contacting an antibody that binds an hB7-H3cell surface receptor or tumor associated antigen expressed on a tumorcell with a drug-linker synthon under conditions in which thedrug-linker synthon covalently links to the antibody through a maleimidemoiety as shown in formulae (IIe) and (IIf), or through an acetyl halideas shown in(IIg), or through a vinyl sulfone as shown in (IIh).

wherein D is the Bcl-xL inhibitor drug according to structural formula(IIa), (IIb), (IIc) or (IId) as described above and L¹ is the portion ofthe linker not formed from the maleimide, acetyl halide or vinyl sulfoneupon attachment of the synthon to the antibody; and wherein thedrug-linker synthon is selected from the group consisting of synthonexamples 2.1 to 2.176 (Table B), or a pharmaceutically acceptable saltthereof.

In certain embodiments, the contacting step is carried out underconditions such that the anti-B7-H3 ADC has a DAR of 2, 3 or 4.

III.B. Anti-B7-H3 ADCs: Other Exemplary Drugs for Conjugation

Anti-B7-H3 antibodies may be used in ADCs to target one or more drug(s)to a cell of interest. e.g., a cancer cell expressing B7-H3. Theanti-B7-H3 ADCs of the invention provide a targeted therapy that may,for example, reduce the side effects often seen with anti-cancertherapies, as the one or more drug(s) is delivered to a specific cell.

Auristatins

Anti-B7-H3 antibodies of the invention, e.g., the huAb13v1, huAb3v2.5,or huAb3v2.6 antibody, may be conjugated to at least one auristatin.Auristatins represent a group of dolastatin analogs that have generallybeen shown to possess anticancer activity by interfering withmicrotubule dynamics and GTP hydrolysis, thereby inhibiting cellulardivision. For example, auristatin E (U.S. Pat. No. 5,635,483) is asynthetic analogue of the marine natural product dolastatin 10, acompound that inhibits tubulin polymerization by binding to the samesite on tubulin as the anticancer drug vincristine (G. R. Pettit. Prog.Chem. Org. Nat. Prod. 70: 1-79 (1997)). Dolastatin 10, auristatin PE,and auristatin E are linear peptides having four amino acids, three ofwhich are unique to the dolastatin class of compounds. Exemplaryembodiments of the auristatin subclass of mitotic inhibitors include,but are not limited to, monomethyl auristatin D (MMAD or auristatin Dderivative), monomethyl auristatin E (MMAE or auristatin E derivative),monomethyl auristatin F (MMAF or auristatin F derivative), auristatin Fphenylenediamine (AFP), auristatin EB (AEB), auristatin EFP (AEFP), and5-benzoylvaleric acid-AE ester (AEVB). The synthesis and structure ofauristatin derivatives are described in U.S. Patent ApplicationPublication Nos. 2003-0083263, 2005-0238649 and 2005-0009751;International Patent Publication No. WO 04/010957. International PatentPublication No. WO 02/088172, and U.S. Pat. Nos. 6,323,315; 6,239,104;6,034,065; 5,780,588 5,665,860; 5,663,149; 5,635,483; 5,599,902;5,554,725; 5,530,097; 5,521,284; 5,504,191; 5,410,024; 5,138,036;5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444; and 4,486,414,each of which is incorporated by reference herein.

In one embodiment, anti-B7-H3 antibodies of the invention. e.g.,huAb13v1, huAb3v2.5, or huAb3v2.6, are conjugated to at least one MMAE(mono-methyl auristatin E). Monomethyl auristatin E (MMAE, vedotin)inhibits cell division by blocking the polymerization of tubulin.However, due to its super toxicity, auristatin E cannot be used as adrug itself. Auristatin E can be linked to a monoclonal antibody (mAb)that recognizes a specific marker expression in cancer cells and directsMMAE to the cancer cells. In one embodiment, the linker linking MMAE tothe anti-B7-H3 antibody is stable in extracellular fluid (i.e., themedium or environment that is external to cells), but is cleaved bycathepsin once the ADC has bound to the specific cancer cell antigen andentered the cancer cell, thus releasing the toxic MMAE and activatingthe potent anti-mitotic mechanism.

In one embodiment, an anti-B7-H3 antibody described herein, e.g.,huAb13v, huAb3v2.5, or huAb3v2.6, is conjugated to at least one MMAF(monomethylauristatin F). Monomethyl auristatin F (MMAF) inhibits celldivision by blocking the polymerization of tubulin. It has a chargedC-terminal phenylalanine residue that attenuates its cytotoxic activitycompared to its uncharged counterpart MMAE. However, due to its supertoxicity, auristatin F cannot be used as a drug itself, but can belinked to a monoclonal antibody (mAb) that directs it to the cancercells. In one embodiment, the linker to the anti-B7-H3 antibody isstable in extracellular fluid, but is cleaved by cathepsin once theconjugate has entered a tumor cell, thus activating the anti-mitoticmechanism.

The structures of MMAF and MMAE are provided below.

An example of huAb13v1, huAb3v2.5, or huAb3v2.6-vcMMAE is also providedin FIG. 3. Notably. FIG. 3 describes a situation where the antibody(e.g. huAb13v1, huAb3v2.5, or huAb3v2.6) is coupled to a single drugand, therefore, has a DAR of 1. In certain embodiments, the ADC willhave a DAR of 2 to 8 or, alternatively. 2 to 4.

Other Drugs for Conjugation

Examples of drugs that may be used in ADCs, i.e, drugs that may beconjugated to the anti-B7-H3 antibodies of the invention, are providedbelow, and include mitotic inhibitors, antitumor antibiotics,immunomodulating agents, gene therapy vectors, alkylating agents,antiangiogenic agents, antimetabolites, boron-containing agents,chemoprotective agents, hormone agents glucocorticoids, photoactivetherapeutic agents, oligonucleotides, radioactive isotopes,radiosensitizers, topoisomerase inhibitors, kinase inhibitors, andcombinations thereof.

1. Mitotic Inhibitors

In one aspect, anti-B7-H3 antibodies may be conjugated to one or moremitotic inhibitor(s) to form an ADC for the treatment of cancer. Theterm “mitotic inhibitor”, as used herein, refers to a cytotoxic and/ortherapeutic agent that blocks mitosis or cell division, a biologicalprocess particularly important to cancer cells. A mitotic inhibitordisrupts microtubules such that cell division is prevented, often byeffecting microtubule polymerization (e.g., inhibiting microtubulepolymerization) or microtubule depolymerization (e.g., stabilizing themicrotubule cytoskeleton against depolymerization). Thus, in oneembodiment, an anti-B7-H3 antibody of the invention is conjugated to oneor more mitotic inhibitor(s) that disrupts microtubule formation byinhibiting tubulin polymerization. In another embodiment, an anti-B7-H3antibody of the invention is conjugated to one or more mitoticinhibitor(s) that stabilizes the microtubule cytoskeleton fromdepolymerization. In one embodiment, the mitotic inhibitor used in theADCs of the invention is Ixempra (ixabepilone). Examples of mitoticinhibitors that may be used in the anti-B7-H3 ADCs of the invention areprovided below. Included in the genus of mitotic inhibitors areauristatins, described above.

a. Dolastatins

The anti-B7-H3 antibodies of the invention may be conjugated to at leastone dolastatin to form an ADC. Dolastatins are short peptidic compoundsisolated from the Indian Ocean sea hare Dolabella auricularia (seePettit et al., J. Am. Chem. Soc., 1976, 98.4677). Examples ofdolastatins include dolastatin 10 and dolastatin 15. Dolastatin 15, aseven-subunit depsipeptide derived from Dolabella auricularia, and is apotent antimitotic agent structurally related to the antitubulin agentdolastatin 10, a five-subunit peptide obtained from the same organism.Thus, in one embodiment, the anti-B7-H3 ADC of the invention comprisesan anti-B7-H3 antibody, as described herein, and at least onedolastatin. Auristatins, described above, are synthetic derivatives ofdolastatin 10.

b. Maytansinoids

The anti-B7-H3 antibodies of the invention may be conjugated to at leastone maytansinoid to form an ADC. Maytansinoids are potent antitumoragents that were originally isolated from members of the higher plantfamilies Celastraceae, Rhamnaceae, and Euphorbiaceae, as well as somespecies of mosses (Kupchan et al, J. Am. Chem. Soc. 94:1354-1356 [1972]:Wani et al. J. Chem. Soc. Chem. Commun. 390: [1973]; Powell et al, J.Nat. Prod. 46:660-666 [1983]; Sakai et al, J. Nat. Prod. 51:845-850[1988]; and Suwanborirux et al, Experientia 46:117-120 [1990]). Evidencesuggests that maytansinoids inhibit mitosis by inhibiting polymerizationof the microtubule protein tubulin, thereby preventing formation ofmicrotubules (see, e.g., U.S. Pat. No. 6,441,163 and Remillard et al.,Science, 189, 1002-1005 (1975)). Maytansinoids have been shown toinhibit tumor cell growth in vitro using cell culture models, and invivo using laboratory animal systems. Moreover, the cytotoxicity ofmaytansinoids is 1,000-fold greater than conventional chemotherapeuticagents, such as, for example, methotrexate, daunorubicin, andvincristine (see, e.g., U.S. Pat. No. 5,208,020).

Maytansinoids to include maytansine, maytansinol, C-3 esters ofmaytansinol, and other maytansinol analogues and derivatives (see, e.g.,U.S. Pat. Nos. 5,208,020 and 6,441,163, each of which is incorporated byreference herein). C-3 esters of maytansinol can be naturally occurringor synthetically derived. Moreover, both naturally occurring andsynthetic C-3 maytansinol esters can be classified as a C-3 ester withsimple carboxylic acids, or a C-3 ester with derivatives ofN-methyl-L-alanine, the latter being more cytotoxic than the former.Synthetic maytansinoid analogues are described in, for example, Kupchanet al., J. Med. Chem., 21, 31-37 (1978).

Suitable maytansinoids for use in ADCs of the invention can be isolatedfrom natural sources, synthetically produced, or semi-syntheticallyproduced. Moreover, the maytansinoid can be modified in any suitablemanner, so long as sufficient cytotoxicity is preserved in the ultimateconjugate molecule. In this regard, maytansinoids lack suitablefunctional groups to which antibodies can be linked. A linking moietydesirably is utilized to link the maytansinoid to the antibody to formthe conjugate, and is described in more detail in the linker sectionbelow. The structure of an exemplary maytansinoid, mertansine (DM1), isprovided below.

Representative examples of maytansinoids include, but are not limited,to DM1 (N^(2′)-deacetyl-N^(2′)-(3-mercapto-1-oxopropyl)-maytansine; alsoreferred to as mertansine, drug maytansinoid 1; ImmunoGen, Inc.; seealso Chari et al. (1992) Cancer Res 52:127). DM2, DM3(N^(2′)-deacetyl-N²-(4-mercapto-1-oxopentyl)-maytansine), DM4(4-methyl-4-mercapto-1-oxopentyl)-maytansine), and maytansinol (asynthetic maytansinoid analog). Other examples of maytansinoids aredescribed in U.S. Pat. No. 8,142,784, incorporated by reference herein.

Ansamitocins are a group of maytansinoid antibiotics that have beenisolated from various bacterial sources. These compounds have potentantitumor activities. Representative examples include, but are notlimited to ansamitocin P1, ansamitocin P2, ansamitocin P3, andansamitocin P4.

In one embodiment of the invention, an anti-B7-H3 antibody is conjugatedto at least one DM. In one embodiment, an anti-B7-H3 antibody isconjugated to at least one DM2. In one embodiment, an anti-B7-H3antibody is conjugated to at least one DM3. In one embodiment, ananti-B7-H3 antibody is conjugated to at least one DM4.

d. Plant Alkaloids

The anti-B7-H3 antibodies of the invention may be conjugated to at leastone plant alkaloid. e.g., a taxane or vinca alkaloid. Plant alkaloidsare chemotherapy treatments derived made from certain types of plants.The vinca alkaloids are made from the periwinkle plant (Catharanthusrosea), whereas the taxanes are made from the bark of the Pacific Yewtree (taus). Both the vinca alkaloids and taxanes are also known asantimicrotubule agents, and are described in more detail below.

Taxanes

Anti-B7-H3 antibodies described herein may be conjugated to at least onetaxane. The term “taxane” as used herein refers to the class ofantineoplastic agents having a mechanism of microtubule action andhaving a structure that includes the taxane ring structure and astereospecific side chain that is required for cytostatic activity. Alsoincluded within the term “taxane” are a variety of known derivatives,including both hydrophilic derivatives, and hydrophobic derivatives.Taxane derivatives include, but not limited to, galactose and mannosederivatives described in International Patent Application No. WO99/18113; piperazino and other derivatives described in WO 99/14209taxane derivatives described in WO 99/09021, WO 98/22451, and U.S. Pat.No. 5,869,680; 6-thio derivatives described in WO 98/28288; sulfenamidederivatives described in U.S. Pat. No. 5,821,263; and taxol derivativedescribed in U.S. Pat. No. 5,415,869, each of which is incorporated byreference herein. Taxane compounds have also previously been describedin U.S. Pat. Nos. 5,641,803, 5,665,671, 5,380,751, 5,728,687, 5,415,869,5,407,683, 5,399,363, 5,424,073, 5,157,049, 5,773,464, 5,821,263,5,840,929, 4,814,470, 5,438,072, 5,403,858, 4,960,790, 5,433,364,4,942,184, 5,362,831, 5,705,503, and 5,278,324, all of which areexpressly incorporated by reference. Further examples of taxanesinclude, but are not limited to, docetaxel (Taxotere; Sanofi Aventis),paclitaxel (Abraxane or Taxol; Abraxis Oncology), carbazitaxel,tesetaxel, opaxio, larotaxel, taxoprexin, BMS-184476, hongdoushan A,hongdoushan B, and hongdoushan C, and nanoparticle paclitaxel (ABI-007Abraxene; Abraxis Bioscience).

In one embodiment, the anti-B7-H3 antibody of the invention isconjugated to at least one docetaxel molecule. In one embodiment, theanti-B7-H3 antibody of the invention is conjugated to at least onepaclitaxel molecule.

Vinca Alkaloids

In one embodiment, the anti-B7-H3 antibody is conjugated to at least onevinca alkaloid. Vinca alkaloids are a class of cell-cycle-specific drugsthat work by inhibiting the ability of cancer cells to divide by actingupon tubulin and preventing the formation of microtubules. Examples ofvinca alkaloids that may be used in the ADCs of the invention include,but are not limited to, vindesine sulfate, vincristine, vinblastine, andvinorelbine.

2. Antitumor Antibiotics

Anti-B7-H3 antibodies of the invention may be conjugated to one or moreantitumor antibiotic(s) for the treatment of cancer. As used herein, theterm “antitumor antibiotic” means an antineoplastic drug that blockscell growth by interfering with DNA and is made from a microorganism.Often, antitumor antibiotics either break up DNA strands or slow down orstop DNA synthesis. Examples of antitumor antibiotics that may beincluded in the anti-B7-H3 ADCs of the invention include, but are notlimited to, actinomycines (e.g., pyrrolo[2,1-c][1,4]benzodiazepines),anthracyclines, calicheamicins, and duocarmycins, described in moredetail below.

a. Actinomycins

The anti-B7-H3 antibodies of the invention may be conjugated to at leastone actinomycin. Actinomycins are a subclass of antitumor antibioticsisolated from bacteria of the genus Streptomyces. Representativeexamples actinomycins include, but are not limited to, actinomycin D(Cosmegen [also known as actinomycin, dactinomycin, actinomycin IV,actinomycin C1], Lundbeck, Inc.), anthramycin, chicamycin A, DC-81,mazethramycin, neothramycin A, neothramycin B, porothramycin,prothracarcin B, SG2285, sibanomicin, sibiromycin, and tomaymycin. Inone embodiment, the anti-B7-H3 antibody of the invention is conjugatedto at least one pyrrolobenzodiazepine (PBD). Examples of PBDs include,but are not limited to, anthramycin, chicamycin A, DC-81, mazethramycin,neothramycin A, neothramycin B, porothramycin, prothracarcin B, SG2000(SJG-136), SG2202 (ZC-207), SG2285 (ZC-423), sibanomicin, sibiromycinand tomaymycin. Thus, in one embodiment, anti-B7-H3 antibodies of theinvention are conjugated to at least one actinomycin. e.g., actinomycinD, or at least one PBD, e.g., a pyrrolobenzodiazepine (PBD) dimer.

The structures of PBDs can be found, for example, in U.S. PatentApplication Pub. Nos. 2013/0028917 and 2013/0028919, and in WO2011/130598 A1, each of which are incorporated herein by reference intheir entirety. The generic structure of a PBD is provided below.

PBDs differ in the number, type and position of substituents, in boththeir aromatic A rings and pyrrolo C rings, and in the degree ofsaturation of the C ring. In the B-ring, there is generally an imine(N═C), a carbinolamine (NH—CH(OH)), or a carbinolamine methyl ether(NH—CH(OMe)) at the N10-C11 position which is the electrophilic centerresponsible for alkylating DNA. All of the known natural products havean (S)-configuration at the chiral C11α position which provides themwith a right-handed twist when viewed from the C ring towards the Aring. The PBD examples provided herein may be conjugated to theanti-B7-H3 antibodies of the invention. Further examples of PBDs whichmay be conjugated to the anti-B7-H3 antibodies of the invention can befound, for example, in U.S. Patent Application Publication Nos.2013/0028917 A1 and 2013/0028919 A1, in U.S. Pat. No. 7,741,319 B2, andin WO 2011/130598 A1 and WO 2006/111759 A1, each of which areincorporated herein by reference in their entirety.

A representative PBD dimer having the following formula XXX may beconjugated to the anti-B7-H3 antibodies of the invention:

wherein:

R³⁰ is of formula XXXI:

where A is a C₅₋₇ aryl group, X is a group conjugated to the Linker unitselected from the group consisting of —O—, —S—, —C(O)O—, —C(O)—,—NH(C═O)—, and —N(R^(N))—, wherein R^(N) is selected from the groupconsisting of H, C₁₋₄alkyl and (C₂H₄O)_(m)CH₃, where s is 1 to 3, andeither:

(i) Q¹ is a single bond, and Q² is selected from the group consisting ofa single bond and —Z—(CH₂)_(n)—, where Z is selected from the groupconsisting of a single bond, O, S and NH and n is from 1 to 3; or

(ii) Q¹ is —CH═CH— and Q² is a single bond.

R¹³⁰ is a C₅₋₁₀ aryl group, optionally substituted by one or moresubstituents selected from the group consisting of halo, nitro, cyano,C₁₋₁₂ alkoxy, C₃₋₂₀ heterocycloalkoxy, C₅₋₂₀ aryloxy, heteroaryloxy,alkylalkoxy, arylalkoxy, alkylaryloxy, heteroarylalkoxy,alkylheteroaryloxy, C₁₋₇-alkyl, C₃₋₇ heterocyclyl andbis-oxy-C₁₋₃alkylene;

-   -   R³¹ and R³³ are independently selected from the group consisting        of H, R^(x), OH, OR^(x), SH, SR^(x), NH₂, NHR^(x), NR^(x)R^(x),        nitro, Me₃Sn and halo;

where R and R′ are independently selected from the group consisting ofoptionally substituted C₁₋₁₂alkyl, C₃₋₂₀ heterocyclyl and C₅₋₂₀-arylgroups:

R³² is selected from the group consisting of H, R^(x), OH, OR^(x), S,SR^(x), NH₂. NHR^(x), NHR^(x)R^(xx), nitro, Me₃Sn and halo;

either:

(a) R³⁴ is H, and R¹¹ is OH, OR^(xA), where R^(xA) is C₁₋₄ alkyl;

(b) R³⁴ and R³⁵ form a nitrogen-carbon double bond between the nitrogenand carbon atoms to which they are bound; or

(c) R³⁴ is H and R³⁵ is SO₂M, where z is 2 or 3;

R^(xxx) is a C₃₋₁₂ alkylene group, which chain may be interrupted by oneor more heteroatoms, selected from the group consisting of O, S, NH, andan aromatic ring;

Y^(x) and Y^(x′) are is selected from the group consisting of O, S, andNH:

R^(31′), R^(=′), R^(33′) are selected from the same groups as R³¹, R³²and R³³ respectively and R^(34′) and R^(35′) are the same as R³⁴ andR³⁵, and each M is a monovalent pharmaceutically acceptable cation orboth M groups together are a divalent pharmaceutically acceptable canon.

C₁₋₁₂ alkyl: The term “C₁₋₁₂ alkyl” as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from a carbonatom of a hydrocarbon compound having from 1 to 12 carbon atoms, whichmay be aliphatic or alicyclic, and which may be saturated or unsaturated(e.g. partially unsaturated, fully unsaturated). Thus, the term “alkyl”includes the sub-classes alkenyl, alkynyl, cycloalkyl, etc., discussedbelow.

Examples of saturated alkyl groups include, but are not limited to,methyl (C₁), ethyl (C₂), propyl (C₃), butyl (C₄), pentyl (C₅), hexyl(C₆) and heptyl (C₇).

Examples of saturated linear alkyl groups include, but are not limitedto, methyl (C₁), ethyl (C₂), n-propyl (C₃), n-butyl (C₄), n-pentyl(amyl) (C₅), n-hexyl (C₆) and n-heptyl (C₇).

Examples of saturated branched alkyl groups include iso-propyl (C₃),iso-butyl (C₄), sec-butyl (C₄), tert-butyl (C₄), iso-pentyl (C₅), andneo-pentyl (C₅).

C₃₋₂₀ heterocyclyl: The term “C₃₋₂₀ heterocyclyl” as used herein,pertains to a monovalent moiety obtained by removing a hydrogen atomfrom a ring atom of a heterocyclic compound, which moiety has from 3 to20 ring atoms, of which from 1 to 10 are ring heteroatoms. Preferably,each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ringheteroatoms.

In this context, the prefixes (e.g. C₃₋₂₀, C₃₋₇, C₅₋₆, etc.) denote thenumber of ring atoms, or range of number of ring atoms, whether carbonatoms or heteroatoms. For example, the term “C₅₋₆ heterocyclyl”, as usedherein, pertains to a heterocyclyl group having 5 or 6 ring atoms.

Examples of monocyclic heterocyclyl groups include, but are not limitedto, those derived from:

N₁: aziridine (C₃), azetidine (C₄), pyrrolidine (tetrahydropyrrole)(C₅), pyrroline (e.g., 3-pyrroline, 2,5-dihydropyrrole) (C₆), 2H-pyrroleor 3H-pyrrole (isopyrrole, isoazole) (C₅), piperidine (C₆),dihydropyridine (C₆), tetrahydropyridine (C₆), azepine (C₇); O₁: oxirane(C₃), oxetane (C₄), oxolane (tetrahydrofuran) (C₅), oxole (dihydrofuran)(C₅), oxane (tetrahydropyran) (C₆), dihydropyran (C₆), pyran (C₆),oxepin (C₇); S₁: thiirane (C₃), thietane (C₄), thiolane(tetrahydrothiophene) (C₅), thiane (tetrahydrothiopyran) (C₆), thiepane(C); O₂: dioxolane (C₆), dioxane (C₆), and dioxepane (C₇); O₃: trioxane(C₆): N₂; imidazolidine (C₅), pyrazolidine (diazolidine) (C₅),imidazoline (C₅), pyrazoline (dihydropyrazole) (C₅), piperazine (C₆);N₁O₁: tetrahydrooxazole (C₅), dihydrooxazole (C₅), tetrahydroisoxazole(C₅), dihydroisoxazole (C₅), morpholine (C₆), tetrahydrooxazine (C₆),dihydrooxazine (C₆), oxazine (C₆); N₁S₁: thiazoline (C₅), thiazolidine(C₆), thiomorpholine (C₆): N_(I)O_(I): oxadiazine (C₆); O₁S₁: oxathiole(C₆) and oxathiane (thioxane) (C₆): and, N₁O₁S₁: oxathiazine (C₆).

Examples of substituted monocyclic heterocyclyl groups include thosederived from saccharides, in cyclic form, for example, furanoses (C₅),such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse,and pyranoses (C₆), such as allopyranose, altropyranose, glucopyranose,mannopyranose, gulopyranose, idopyranose, galactopyranose, andtalopyranose.

C₅₋₂₀ aryl: The term “C₅₋₂₀ aryl” as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from an aromaticring atom of an aromatic compound, which moiety has from 3 to 20 ringatoms. Preferably, each ring has from 5 to 7 ring atoms.

In this context, the prefixes (e.g. C₃₋₂₀, C₅₋₇, C₅₋₆, etc.) denote thenumber of ring atoms, or range of number of ring atoms, whether carbonatoms or heteroatoms. For example, the term “C₅₋₆ aryl” as used herein,pertains to an aryl group having 5 or 6 ring atoms.

In one embodiment, the anti-B7-H3 antibodies of the invention may beconjugated to a PBD dimer having the following formula XXXIa:

wherein the above structure describes the PBD dimer SG2202 (ZC-207) andis conjugated to the anti-B7-H3 antibody of the invention via a linkerL. SG2202 (ZC-207) is disclosed in, for example, U.S. Patent App. Pub.No. 2007/0173497, which is incorporated herein by reference in itsentirety.

In another embodiment, a PBD dimer, SGD-1882, is conjugated toanti-B7-H3 antibody of the invention via a drug linker, as depicted inFIG. 4. SGD-1882 is disclosed in Sutherland et al. (2013) Blood122(8):1455 and in U.S Patent App. Pub. No. 2013/0028919, which isincorporated herein by reference in its entirety. As described in FIG.4, the PBD dimer SGD-1882 may be conjugated to an antibody via anmc-val-ala-dipeptide linker (collectively referred to as SGD-1910 inFIG. 4). In a certain embodiment, an anti-B7-H3 antibody, as disclosedherein, is conjugated to the PBD dimer described in FIG. 4. Thus, in afurther embodiment, the invention includes an anti-B7-H3 antibody, asdisclosed herein, conjugated to a PBD dimer via a mc-val-ala-dipeptidelinker, as described in FIG. 4. In certain embodiments, the inventionincludes an anti-B7-H3 antibody comprising a heavy chain variable regioncomprising a CDR3 domain comprising the amino acid sequence of SEQ IDNO: 35, a CDR2 domain comprising the amino acid sequence of SEQ ID NO:34, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO:33, and a light chain variable region comprising a CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 39, a CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 38, and a CDR1 domaincomprising the amino acid sequence of SEQ ID NO: 37, conjugated to aPBD, including, but not limited to, the PBD dimer described in FIG. 4.In certain embodiments, the invention includes an anti-B7-H3 antibodycomprising a heavy chain variable region comprising a CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 12, a CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 140, and a CDR1 domaincomprising the amino acid sequence of SEQ ID NO: 10, and a light chainvariable region comprising a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 15, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 7, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 136, conjugated to a PBD, including, but notlimited to, the PBD dimer described in FIG. 4. In certain embodiments,the invention includes an anti-B7-H3 antibody comprising a heavy chainvariable region comprising a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 12, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 140, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 10, and a light chain variable region comprisinga CDR3 domain comprising the amino acid sequence of SEQ ID NO: 15, aCDR2 domain comprising the amino acid sequence of SEQ ID NO: 7, and aCDR1 domain comprising the amino acid sequence of SEQ ID NO: 138,conjugated to a PBD, including, but not limited to, the PBD dimerdescribed in FIG. 4. In certain embodiments, the invention includes ananti-B7-H3 antibody comprising the heavy chain variable region ofhuAb13v1 as defined by the amino acid sequence set forth in SEQ ID NO:147, or huAb3v2.5 or huAb3v2.6 as defined by the amino acid sequence setforth in SEQ ID NO: 139, and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 144, 135, or 137 corresponding tohuAb13v1, huAb3v2.5, or huAb3v2.6, respectively, wherein the antibody isconjugated to a PBD, such as, but not limited to, the exemplary PBDdimer of FIG. 4.

b. Anthracyclines

Anti-B7-H3 antibodies of the invention may be conjugated to at least oneanthracycline.

Anthracyclines are a subclass of antitumor antibiotics isolated frombacteria of the genus Streptomyces. Representative examples include, butare not limited to daunorubicin (Cerubidine, Bedford Laboratories),doxorubicin (Adriamycin, Bedford Laboratories; also referred to asdoxorubicin hydrochloride, hydroxydaunorubicin, and Rubex), epirubicin(Ellence, Pfizer), and idarubicin (Idamycin; Pfizer Inc.). Thus, in oneembodiment, the anti-B7-H3 antibody of the invention is conjugated to atleast one anthracycline, e.g., doxorubicin.

c. Calicheamicins

The anti-B7-H3 antibodies of the invention may be conjugated to at leastone calicheamicin.

Calicheamicins are a family of enediyne antibiotics derived from thesoil organism Micromonospora echinospora. Calicheamicins bind the minorgroove of DNA and induce double-stranded DNA breaks, resulting in celldeath with a 100 fold increase over other chemotherapeutics (Damle etal. (2003) Curr Opin Pharmacol 3:386). Preparation of calicheamicinsthat may be used as drug conjugates in the invention have beendescribed, see U.S. Pat. Nos. 5,712,374; 5,714,586; 5,739,116;5,767,285; 5,770,701; 5,770,710; 5,773,001; and 5,877,2%. Structuralanalogues of calicheamicin which may be used include, but are notlimited to, γ₁ ^(l), α₂ ^(l), α₃ ^(l), N-acetyl-γ₁ ^(l), PSAG and θ¹_(l) (Himnan et al., Cancer Research 53:3336-3342 (1993), Lode et al.,Cancer Research 58:2925-2928 (1998) and the aforementioned U.S. Pat.Nos. 5,712,374; 5,714,586; 5,739,116; 5,767,285; 5,770,701; 5,770,710;5,773,001; and 5,877,296). Thus, in one embodiment, the anti-B7-H3antibody of the invention is conjugated to at least one calicheamicin.

d. Duocarmycins

Anti-B7-H3 antibodies of the invention may be conjugated to at least oneduocarmycin. Duocarmycins are a subclass of antitumor antibioticsisolated from bacteria of the genus Streptomyces. (see Nagamura andSaito (1998) Chemistry of Hetermcyclic Compounds, Vol. 34, No. 12).Duocarmycins bind to the minor groove of DNA and alkylate the nucleobaseadenine at the N3 position (Boger (1993) Pure and Appl Chem 65(6):1123;and Boger and Johnson (1995) PNAS USA 92:3642). Synthetic analogs ofduocarmycins include, but are not limited to, adozelesin, bizelesin, andcarzelesin. Thus, in one embodiment, the anti-B7-H3 antibody of theinvention is conjugated to at least one duocarmycin.

e. Other Antitumor Antibiotics

In addition to the foregoing, additional antitumor antibiotics that maybe used in the anti-B7-H3 ADCs of the invention include bleomycin(Blenoxane, Bristol-Myers Squibb), mitomycin, and plicamycin (also knownas mithramycin).

3. Immunomodulating Agents

In one aspect, anti-B7-H3 antibodies of the invention may be conjugatedto at least one immunomodulating agent. As used herein, the term“immunomodulating agent” refers to an agent that can stimulate or modifyan immune response. In one embodiment, an immunomodulating agent is animmunostimulator that enhances a subject's immune response. In anotherembodiment, an immunomodulating agent is an immunosuppressant thatprevents or decreases a subject's immune response. An immunomodulatingagent may modulate myeloid cells (monocytes, macrophages, dendriticcells, megakaryocytes and granulocytes) or lymphoid cells (T cells, Bcells and natural killer (NK) cells) and any further differentiated cellthereof. Representative examples include, but are not limited to,bacillus calmette-guerin (BCG) and levamisole (Ergamisol). Otherexamples of immunomodulating agents that may be used in the ADCs of theinvention include, but are not limited to, cancer vaccines, cytokines,and immunomodulating gene therapy.

a. Cancer Vaccines

Anti-B7-H3 antibodies of the invention may be conjugated to a cancervaccine. As used herein, the term “cancer vaccine” refers to acomposition (e.g., a tumor antigen and a cytokine) that elicits atumor-specific immune response. The response is elicited from thesubject's own immune system by administering the cancer vaccine, or, inthe case of the instant invention, administering an ADC comprising ananti-B7-H3 antibody and a cancer vaccine. In preferred embodiments, theimmune response results in the eradication of tumor cells in the body(e.g., primary or metastatic tumor cells). The use of cancer vaccinesgenerally involves the administration of a particular antigen or groupof antigens that are, for example, present on the surface a particularcancer cell, or present on the surface of a particular infectious agentshown to facilitate cancer formation. In some embodiments, the use ofcancer vaccines is for prophylactic purposes, while in otherembodiments, the use is for therapeutic purposes. Non-limiting examplesof cancer vaccines that may be used in the anti-B7-H3 ADCs of theinvention include, recombinant bivalent human papillomavirus (HPV)vaccine types 16 and 18 vaccine (Cervarix, GlaxoSmithKline), recombinantquadrivalent human papillomavirus (HPV) types 6, 11, 16, and 18 vaccine(Gardasil, Merck & Company), and sipuleucel-T (Provenge, Dendreon).Thus, in one embodiment, the anti-B7-H3 antibody of the invention isconjugated to at least one cancer vaccine that is either animmunostimulator or is an immunosuppressant.

b. Cytokines

The anti-B7-H3 antibodies of the invention may be conjugated to at leastone cytokine. The term “cytokine” generally refers to proteins releasedby one cell population which act on another cell as intercellularmediators. Cytokines directly stimulate immune effector cells andstromal cells at the tumor site and enhance tumor cell recognition bycytotoxic effector cells (Lee and Margolin (2011) Cancers 3:3856).Numerous animal tumor model studies have demonstrated that cytokineshave broad anti-tumor activity and this has been translated into anumber of cytokine-based approaches for cancer therapy (Lee and Margoli,supra). Recent years have seen a number of cytokines, including GM-CSF,IL-7, IL-12, IL-15, IL-18 and IL-21, enter clinical trials for patientswith advanced cancer (Lee and Margoli, supra).

Examples of cytokines that may be used in the ADCs of the inventioninclude, but are not limited to, parathyroid hormone; thyroxine;insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such asfollicle stimulating hormone (FSH), thyroid stimulating hormone (TSH),and luteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF; platelet-growthfactor; transforming growth factors (TGFs); insulin-like growth factor-Iand -II; erythropoietin (EPO); osteoinductive factors; interferons suchas interferon α, β,and γ, colony stimulating factors (CSFs);granulocyte-macrophage-C-SF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-11, IL-12; tumor necrosis factor; and otherpolypeptide factors including LIF and kit ligand (KL). As used herein,the term cytokine includes proteins from natural sources or fromrecombinant cell culture and biologically active equivalents of thenative sequence cytokincs. Thus, in one embodiment, the inventionprovides an ADC comprising an anti-B7-H3 antibody described herein and acytokine.

c. Colony-Stimulating Factors (CSFs)

The anti-B7-H3 antibodies of the invention may be conjugated to at leastone colony stimulating factor (CSF). Colony stimulating factors (CSFs)are growth factors that assist the bone marrow in making white bloodcells. Some cancer treatments (e.g., chemotherapy) can affect whiteblood cells (which help fight infection); therefore, colony-stimulatingfactors may be introduced to help support white blood cell levels andstrengthen the immune system. Colony-stimulating factors may also beused following a bone marrow transplant to help the new marrow startproducing white blood cells. Representative examples of CSFs that may beused in the anti-B7-H3 ADCs of the invention include, but are notlimited to erythropoietin (Epoetin), filgrastim (Neopogen (also known asgranulocyte colony-stimulating factor (G-CSF); Amgen. Inc.),sargramostim (leukine (granulocyte-macrophage colony-stimulating factorand GM-CSF); Genzyme Corporation), promegapoietin, and Oprelvekin(recombinant IL-11; Pfizer. Inc.). Thus, in one embodiment, theinvention provides an ADC comprising an anti-B7-H3 antibody describedherein and a CSF.

4. Gene Therapy

The anti-B7-H3 antibody of the invention may be conjugated to at leastone nucleic acid (directly or indirectly via a carrier) for genetherapy. Gene therapy generally refers to the introduction of geneticmaterial into a cell whereby the genetic material is designed to treat adisease. As it pertains to immunomodulatory agents, gene therapy is usedto stimulate a subject's natural ability to inhibit cancer cellproliferation or kill cancer cells. In one embodiment, the anti-B7-H3ADC of the invention comprises a nucleic acid encoding a functional,therapeutic gene that is used to replace a mutated or otherwisedysfunctional (e.g. truncated) gene associated with cancer. In otherembodiments, the anti-B7-H3 ADC of the invention comprises a nucleicacid that encodes for or otherwise provides for the production of atherapeutic protein to treat cancer. The nucleic acid that encodes thetherapeutic gene may be directly conjugated to the anti-B7-H3 antibody,or alternatively, may be conjugated to the anti-B7-H3 antibody through acarrier. Examples of carriers that may be used to deliver a nucleic acidfor gene therapy include, but are not limited to, viral vectors orliposomes.

5. Alkylating Agents

The anti-B7-H3 antibodies of the invention may be conjugated to one ormore alkylating agent(s). Alkylating agents are a class ofantineoplastic compounds that attaches an alkyl group to DNA. Examplesof alkylating agents that may be used in the ADCs of the inventioninclude, but are not limited to, alkyl sulfonates, ethylenimimes,methylamine derivatives, epoxides, nitrogen mustards, nitrosoureas,triazines, and hydrazines.

a. Alkyl Sulfonates

The anti-B7-H3 antibodies of the invention may be conjugated to at leastone alkyl sulfonate. Alkyl sulfonates are a subclass of alkylatingagents with a general formula: R—SO₂—O—R¹, wherein R and R¹ aretypically alkyl or aryl groups. A representative example of an alkylsulfonate includes, but is not limited to, busulfan (Myleran,GlaxoSmithKline; Busulfex IV, PDL BioPharma, Inc.).

b. Nitrogen Mustards

The anti-B7-H3 antibodies of the invention may be conjugated to at leastone nitrogen mustard. Representative examples of this subclass ofanti-cancer compounds include, but are not limited to chlorambucil(Leukeran, GlaxoSmithKline), cyclophosphamide (Cytoxan, Bristol-MyersSquibb; Neosar. Pfizer, Inc.), estramustine (estramustine phosphatesodium or Estracyt), Pfizer, Inc.), ifosfamide (Ifex, Bristol-MyersSquibb), mechlorethamine (Mustargen, Lundbeck Inc.), and melphalan(Alkeran or L-Pam or phenylalanine mustard; GlaxoSmithKline).

c. Nitrosoureas

The anti-B7-H3 antibody of the invention may be conjugated to at leastone nitrosourea.

Nitrosoureas are a subclass of alkylating agents that are lipid soluble.Representative examples include, but are not limited to, carmustine(BCNU [also known as BiCNU, N,N-Bis(2-chloroethyl)-N-nitrosourea, or 1,3-bis (2-chloroethyl)-1-nitrosourea], Bristol-Myers Squibb), fotemustine(also known as Muphoran), lomustine (CCNU or1-(2-chloro-ethyl)-3-cyclohexyl-1-nitrosourea. Bristol-Myers Squibb),nimustine (also known as ACNU), and streptozocin (Zanosar, TevaPharmaceuticals).

d. Triazines and Hydrazines

The anti-B7-H3 antibody of the invention may be conjugated to at leastone triazine or hydrazine. Triazines and hydrazines are a subclass ofnitrogen-containing alkylating agents. In some embodiments, thesecompounds spontaneously decompose or can be metabolized to produce alkyldiazonium intermediates that facilitate the transfer of an alkyl groupto nucleic acids, peptides, and/or polypeptides, thereby causingmutagenic, carcinogenic, or cytotoxic effects. Representative examplesinclude, but are not limited to dacarbazine (DTIC-Dome, Bayer HealthcarePharmaceuticals Inc.), procarbazine (Mutalane, Sigma-TauPharmaceuticals. Inc.), and temozolomide (Temodar, Schering Plough).

e. Other Alkylating Agents

The anti-B7-H3 antibodies of the invention may be conjugated to at leastone ethylenimine, methylamine derivative, or epoxide. Ethylenimines area subclass of alkylating agents that typically containing at least oneaziridine ring. Epoxides represent a subclass of alkylating agents thatare characterized as cyclic ethers with only three ring atoms.

Representatives examples of ethylenimines include, but are not limitedto thiopeta (Thioplex, Amgen), diaziquone (also known as aziridinylbenzoquinone (AZQ)), and mitomycin C. Mitomycin C is a natural productthat contains an aziridine ring and appears to induce cytoxicity throughcross-linking DNA (Dorr R T, et al. Cancer Res. 1985; 45:3510; Kennedy KA. et al Cancer Res. 1985; 45:3541). Representative examples ofmethylamine derivatives and their analogs include, but are not limitedto, altretamine (Hexalen, MGI Pharma, Inc.), which is also known ashexamethylamine and hexastat. Representative examples of epoxides ofthis class of anti-cancer compound include, but are not limited todianhydrogalactitol. Dianhydrogalactitol (1,2:5,6-dianhydrodulcitol) ischemically related to the aziridines and generally facilitate thetransfer of an alkyl group through a similar mechanism as describedabove. Dibromodulcitol is hydrolyzed to dianhydrogalactitol and thus isa pro-drug to an epoxide (Sellei C, et al. Cancer Chemother Rep. 1969;53:377).

6 Antiangiogenic Agents

In one aspect, the anti-B7-H3 antibodies described herein are conjugatedto at least one antiangiogenic agent. Antiangiogenic agents inhibit thegrowth of new blood vessels. Antiangiogenic agents exert their effectsin a variety of ways. In some embodiments, these agents interfere withthe ability of a growth factor to reach its target. For example,vascular endothelial growth factor (VEGF) is one of the primary proteinsinvolved in initiating angiogenesis by binding to particular receptorson a cell surface. Thus, certain antiangiogenic agents, that prevent theinteraction of VEGF with its cognate receptor, prevent VEGF frominitiating angiogenesis. In other embodiments, these agents interferewith intracellular signaling cascades. For example, once a particularreceptor on a cell surface has been triggered, a cascade of otherchemical signals is initiated to promote the growth of blood vessels.Thus, certain enzymes, for example, some tyrosine kinases, that areknown to facilitate intracellular signaling cascades that contribute to,for example, cell proliferation, are targets for cancer treatment. Inother embodiments, these agents interfere with intercellular signalingcascades. Yet, in other embodiments, these agents disable specifictargets that activate and promote cell growth or by directly interferingwith the growth of blood vessel cells. Angiogenesis inhibitoryproperties have been discovered in more than 300 substances withnumerous direct and indirect inhibitory effects.

Representative examples of antiangiogenic agents that may be used in theADCs of the invention include, but are not limited to, angiostatin, ABXEGF, C1-1033, PK-166, EGF vaccine, EKB-569, GW2016, ICR-62, EMD 55900,CP358, PD153035, AG1478, IMC-C225 (Erbitux. ZD1839 (Iressa), OSI-774,Erlotinib (tarceva), angiostatin, arrestin, endostatin. BAY 12-9566 andw/fluorouracil or doxorubicin, canstatin, carboxyamidotriozole and withpaclitaxel, EMD121974, S-24, vitaxin, dimethylxanthenone acetic acid,IM862, lntrleukin-12. Interleukin-2, NM-3, HuMV833. PTK787, RhuMab,angiozyme (ribozyme), IMC-1C11, Neovastat, marimstat, prinomastat,BMS-275291,COL-3, MM1270, SU101, SU6668, SU11248, SU5416, withpaclitaxel, with gemcitabine and cisplatin, and with irinotecan andcisplatin and with radiation, tecogalan, temozolomide and PEG interferonα2b, tetrathiomolybdate, TNP-470, thalidomide, CC-5013 and withtaxotere, tumstatin, 2-methoxyestradiol, VEGF trap, mTOR inhibitors(deforolimus, everolimus (Afinitor, Novartis PharmaceuticalCorporation), and temsirolimus (Torisel, Pfizer, Inc.)), kinaseinhibitors (e.g., erlotinib (Tarceva, Genentech. Inc.), imatinib(Gleevec, Novartis Pharmaceutical Corporation), gefitinib (Iressa,AstraZeneca Pharmaceuticals), dasatinib (Sprycel, Brystol-Myers Squibb),sunitinib (Sutent, Pfizer, Inc.), nilotinib (Tasigna, NovartisPharmaceutical Corporation), lapatinib (Tykerb, GlaxoSmithKlinePharmaceuticals), sorafenib (Nexavar, Bayer and Onyx), phosphoinositide3-kinases (PI3K). Osimertinib, Cobimetinib, Trametinib, Dabrafenib,Dinaciclib).

7. Antimetabolites

The anti-B7-H3 antibodies of the invention may be conjugated to at leastone antimetabolite. Antimetabolites are types of chemotherapy treatmentsthat are very similar to normal substances within the cell. When thecells incorporate an antimetabolite into the cellular metabolism, theresult is negative for the cell, e.g., the cell is unable to divide.Antimetabolites are classified according to the substances with whichthey interfere. Examples of antimetabolites that may be used in the ADCsof the invention include, but are not limited to, a folic acidantagonist (e.g., methotrexate), a pyrimidine antagonist (e.g.,5-Fluorouracil, Foxuridine, Cytarabine, Capecitabine, and Gemcitabine),a purine antagonist (e.g., 6-Mercaptopurine and 6-Thioguanine) and anadenosine deaminase inhibitor (e.g., Cladribine, Fludarabine, Nelarabineand Pentostatin), as described in more detail below.

a. Antifolates

The anti-B7-H3 antibodies of the invention may be conjugated to at leastone antifolate. Antifolates are a subclass of antimetabolites that arestructurally similar to folate. Representative examples include, but arenot limited to, methotrexate, 4-amino-folic acid (also known asaminopterin and 4-aminopteroic acid), lometrexol (LMTX), pemetrexed(Alimpta, Eli Lilly and Company), and trimetrexate (Neutrexin, Ben VenueLaboratories. Inc.)

b. Purine Antagonists

The anti-B7-H3 antibodies of the invention may be conjugated to at leastone purine antagonist. Purine analogs are a subclass of antimetabolitesthat are structurally similar to the group of compounds known aspurines. Representative examples of purine antagonists include, but arenot limited to, azathioprine (Azasan, Salix; Imuran, GlaxoSmithKline),cladribine (Leustatin [also known as 2-CdA], Janssen Biotech. Inc.),mercaptopurine (Purinethol [also known as 6-mercaptoethanol],GlaxoSmithKline), fludarabine (Fludara, Genzyme Corporation),pentostatin (Nipent, also known as 2′-deoxycoformycin (DCF)),6-thioguanine (Lanvis [also known as thioguanine], GlaxoSmithKline).

c. Pyrimidine Antagonists

The anti-B7-H3 antibodies of the invention may be conjugated to at leastone pyrimidine antagonist. Pyrimidine antagonists are a subclass ofantimetabolites that are structurally similar to the group of compoundsknown as purines. Representative examples of pyrimidine antagonistsinclude, but are not limited to azacitidine (Vidaza, CelgeneCorporation), capecitabine (Xeloda, Roche Laboratories), Cytarabine(also known as cytosine arabinoside and arabinosylcytosine, BedfordLaboratories), decitabine (Dacogen, Eisai Pharmaceuticals),5-fluorouracil (Adrucil, Teva Pharmaceuticals; Efudex, ValeantPharmaceuticals, Inc). 5-fluoro-2′-deoxyuridine 5′-phosphate (FdUMP),5-fluorouridine triphosphate, and gemcitabine (Gemzar, Eli Lilly andCompany).

8. Boron-Containing Agents

The anti-B7-H3 antibody of the invention may be conjugated to at leastone boron containing agent. Boron-containing agents comprise a class ofcancer therapeutic compounds which interfere with cell proliferation.Representative examples of boron containing agents include, but are notlimited, to borophycin and bortezomib (Velcade, MilleniumPharmaceuticals).

9. Chemoprotective Agents

The anti-B7-H3 antibodies of the invention may be conjugated to at leastone chemoprotective agent. Chemoprotective drugs are a class ofcompounds, which help protect the body against specific toxic effects ofchemotherapy. Chemoprotective agents may be administered with variouschemotherapies in order to protect healthy cells from the toxic effectsof chemotherapy drugs, while simultaneously allowing the cancer cells tobe treated with the administered chemotherapeutic. Representativechemoprotective agents include, but are not limited to amifostine(Ethyol, Medimmune, Inc.), which is used to reduce renal toxicityassociated with cumulative doses of cisplatin, dexrazoxane (Totect,Apricus Pharma; Zinecard), for the treatment of extravasation caused bythe administration of anthracycline (Totect), and for the treatment ofcardiac-related complications caused by the administration of theantitumor antibiotic doxorubicin (Zinecard), and mesna (Mesnex,Bristol-Myers Squibb), which is used to prevent hemorrhagic cystitisduring chemotherapy treatment with ifocfamide.

10. Hormone Agents

The anti-B7-H3 antibody of the invention may be conjugated to at leastone hormone agent. A hormone agent (including synthetic hormones) is acompound that interferes with the production or activity of endogenouslyproduced hormones of the endocrine system. In some embodiments, thesecompounds interfere with cell growth or produce a cytotoxic effect.Non-limiting examples include androgens, estrogens, medroxyprogesteroneacetate (Provera, Pfizer. Inc.), and progestins.

11. Antihormone Agents

The anti-B7-H3 antibodies of the invention may be conjugated to at leastone antihormone agent. An “antihormone” agent is an agent thatsuppresses the production of and/or prevents the function of certainendogenous hormones. In one embodiment, the antihormone agent interfereswith the activity of a hormone selected from the group comprisingandrogens, estrogens, progesterone, and goanadotropin-releasing hormone,thereby interfering with the growth of various cancer cells.Representative examples of antihormone agents include, but are notlimited to, aminoglutethimide, anastrozole (Arimidex, AstraZenecaPharmaceuticals), bicalutamide (Casodex, AstraZeneca Pharmaceuticals),cyproterone acetate (Cyprostat, Bayer PLC), degarelix (Firmagon, FerringPharmaceuticals), exemestane (Aromasin, Pfizer Inc.), flutamide(Drogenil, Schering-Plough Ltd), fulvestrant (Faslodex, AstraZenecaPharmaceuticals), goserelin (Zolodex, AstraZeneca Pharmaceuticals),letrozole (Femara, Novartis Pharmaceuticals Corporation), leuprolide(Prostap), lupron, medroxyprogesterone acetate (Provera, Pfizer Inc.),Megestrol acetate (Megace, Bristol-Myers Squibb Company), tamoxifen(Nolvadex, AstraZeneca Pharmaceuticals), and triptorelin (Decapetyl,Ferring).

12. Corticosteroids

The anti-B7-H3 antibodies of the invention may be conjugated to at leastone corticosteroid. Corticosteroids may be used in the ADCs of theinvention to decrease inflammation. An example of a corticosteroidincludes, but is not limited to, a glucocorticoid, for example,prednisone (Deltasone, Pharmacia & Upjohn Company, a division of Pfizer,Inc.).

13. Photoactive Therapeutic Agents

The anti-B7-H3 antibodies of the invention may be conjugated to at leastone photoactive therapeutic agent. Photoactive therapeutic agentsinclude compounds that can be deployed to kill treated cells uponexposure to electromagnetic radiation of a particular wavelength.Therapeutically relevant compounds absorb electromagnetic radiation atwavelengths which penetrate tissue. In preferred embodiments, thecompound is administered in a non-toxic form that is capable ofproducing a photochemical effect that is toxic to cells or tissue uponsufficient activation. In other preferred embodiments, these compoundsare retained by cancerous tissue and are readily cleared from normaltissues. Non-limiting examples include various chromagens and dyes.

14. Oligonucleotides

The anti-B7-H3 antibodies of the invention may be conjugated to at leastone oligonucleotide. Oligonucleotides are made of short nucleic acidchains that work by interfering with the processing of geneticinformation. In some embodiments, the oligonucleotides for use in ADCsare unmodified single-stranded and/or double-stranded DNA or RNAmolecules, while in other embodiments, these therapeuticoligonucleotides are chemically-modified single-stranded and/ordouble-stranded DNA or RNA molecules. In one embodiment, theoligonulceotides used in the ADCs are relatively short (19-25nucleotides) and hybridize to a unique nucleic acid sequence in thetotal pool of nucleic acid targets present in cells. Some of theimportant oligonucleotide technologies include the antisenseoligonucleotides (including RNA interference (RNAi)), aptamers, CpGoligonucleotides, and ribozymes.

a. Antisense Oligonucleotides

The anti-B7-H3 antibody of the invention may be conjugated to at leastone antisense oligonucleotide. Antisense oligonucleotides are designedto bind to RNA through Watson-Crick hybridization. In some embodimentsthe antisense oligonucleotide is complementary to a nucleotide encodinga region, domain, portion, or segment of B7-H3. In some embodiments, theantisense oligonucleotide comprises from about 5 to about 100nucleotides, from about 10 to about 50 nucleotides, from about 12 toabout 35, and from about 18 to about 25 nucleotides. In someembodiments, the oligonucleotide is at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or at least 100% homologous to aregion, portion, domain, or segment of the B7-H3 gene. In someembodiments there is substantial sequence homology over at least 15, 20,25, 30, 35, 40, 50, or 100 consecutive nucleotides of the B7-H3 gene. Inpreferred embodiments, the size of these antisense oligonucleotidesranges from 12 to 25 nucleotides in length, with the majority ofantisense oligonucleotides being 18 to 21 nucleotides in length. Thereare multiple mechanisms that can be exploited to inhibit the function ofthe RNA once the oligonucleotide binds to the target RNA (Crooke S T.(1999). Biochim. Biophys. Acta, 1489, 30-42). The best-characterizedantisense mechanism results in cleavage of the targeted RNA byendogenous cellular nucleases, such as RNase H or the nucleaseassociated with the RNA interference mechanism. However,oligonucleotides that inhibit expression of the target gene bynon-catalytic mechanisms, such as modulation of splicing or translationarrest, can also be potent and selective modulators of gene function.

Another RNase-dependent antisense mechanism that has recently receivedmuch attention is RNAi (Fire et al. (1998). Nature, 391, 806-811.;Zamore P D. (2002). Science, 296, 1265-1269.). RNA interference (RNAi)is a post-transcriptional process where a double stranded RNA inhibitsgene expression in a sequence specific fashion. In some embodiments, theRNAi effect is achieved through the introduction of relatively longerdouble-stranded RNA (dsRNA), while in preferred embodiments, this RNAieffect is achieved by the introduction of shorter double-stranded RNAs.e.g. small interfering RNA (siRNA) and/or microRNA (miRNA). In yetanother embodiment. RNAi can also be achieved by introducing of plasmidthat generate dsRNA complementary to target gene. In each of theforegoing embodiments, the double-stranded RNA is designed to interferewith the gene expression of a particular the target sequence withincells. Generally, the mechanism involves conversion of dsRNA into shortRNAs that direct ribonucleases to homologous mRNA targets (summarized,Ruvkun, Science 2294:797 (2001)), which then degrades the correspondingendogenous mRNA, thereby resulting in the modulation of gene expression.Notably, dsRNA has been reported to have anti-proliferative properties,which makes it possible also to envisage therapeutic applications (Aubelet al., Proc. Natl. Acad Sci., USA 88:906 (1991)). For example,synthetic dsRNA has been shown to inhibit tumor growth in mice (Levy etal. Proc. Nat. Acad. Sci. USA, 62:357-361 (1969)), is active in thetreatment of leukemic mice (Zeleznick et al., Proc. Soc. Exp. Biol. Med130:126-128 (1969)), and inhibits chemically induced tumorigenesis inmouse skin (Gelboin et al., Science 167:205-207 (1970)). Thus, in apreferred embodiment, the invention provides for the use of antisenseoligonucleotides in ADCs for the treatment of breast cancer. In otherembodiments, the invention provides compositions and methods forinitiating antisense oligonucleotide treatment, wherein dsRNA interfereswith target cell expression of B7-H3 at the mRNA level, dsRNA, as usedabove, refers to naturally-occurring RNA, partially purified RNA,recombinantly produced RNA, synthetic RNA, as well as altered RNA thatdiffers from naturally-occurring RNA by the inclusion of non-standardnucleotides, non-nucleotide material, nucleotide analogs (e.g. lockednucleic acid (LNA)), deoxyribonucleotides, and any combination thereof.RNA of the invention need only be sufficiently similar to natural RNAthat it has the ability to mediate the antisense oligonucleotide-basedmodulation described herein.

b. Aptamers

The anti-B7-H3 antibodies of the invention may be conjugated to at leastone aptamer. An aptamer is a nucleic acid molecule that has beenselected from random pools based on its ability to bind other molecules.Like antibodies, aptamers can bind target molecules with extraordinaryaffinity and specificity. In many embodiments, aptamers assume complex,sequence-dependent, three-dimensional shapes that allow them to interactwith a target protein, resulting in a tightly bound complex analogous toan antibody-antigen interaction, thereby interfering with the functionof said protein. The particular capacity of aptamers to bind tightly andspecifically to their target protein underlines their potential astargeted molecular therapies.

c. CpG Oligonucleotides

The anti-B7-H3 antibodies of the invention may be conjugated to at leastone CpG oligonucleotide. Bacterial and viral DNA are known to be astrong activators of both the innate and specific immunity in humans.These immunologic characteristics have been associated with unmethylatedCpG dinucleotide motifs found in bacterial DNA. Owing to the fact thatthese motifs are rare in humans, the human immune system has evolved theability to recognize these motifs as an early indication of infectionand subsequently initiate immune responses. Therefore, oligonucleotidescontaining this CpG motif can be exploited to initiate an antitumorimmune response.

d. Ribozymes

The anti-B7-H3 antibody of the invention may be conjugated to at leastone ribozyme. Ribozymes are catalytic RNA molecules ranging from about40 to 155 nucleotides in length. The ability of ribozymes to recognizeand cut specific RNA molecules makes them potential candidates fortherapeutics. A representative example includes angiozyme.

15. Radionuclide Agents (Radioactive Isotopes)

The anti-B7-H3 antibodies of the invention may be conjugated to at leastone radionuclide agent. Radionuclide agents comprise agents that arecharacterized by an unstable nucleus that is capable of undergoingradioactive decay. The basis for successful radionuclide treatmentdepends on sufficient concentration and prolonged retention of theradionuclide by the cancer cell. Other factors to consider include theradionuclide half-life, the energy of the emitted particles, and themaximum range that the emitted particle can travel. In preferredembodiments, the therapeutic agent is a radionuclide selected from thegroup consisting of ¹¹¹In, ¹¹⁷Lu, ²¹²Bi, ²¹³Bi, ²¹¹At, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu,⁹⁰Y, ¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag, ⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb,¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb, ²²³Ra, ²²⁵Ac, ⁵⁹Fe, ⁷⁵Se,⁷⁷As, ⁸⁹Sr, ⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au,¹⁹⁹Au, and ²¹¹Pb. Also preferred are radionuclides that substantiallydecay with Auger-emitting particles. For example, Co-58, Ga-67, Br-80m,Tc-99m, Rh-103m, Pt-109,In-111 1, Sb-119, I-125, Ho-161, Os-189m andIr-192. Decay energies of useful beta-particle-emitting nuclides arepreferably Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-21 1,Ac-225, Fr-221, At-217, Bi-213 and Fm-255. Decay energies of usefulalpha-particle-emitting radionuclides are preferably 2,000-10,000 keV,more preferably 3,000-8,000 keV, and most preferably 4,000-7,000 keV.Additional potential radioisotopes of use include ¹¹C, ¹³N, ¹⁵O, ⁷⁸Br,¹⁹⁸Au, ²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br, ^(113m)In, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru, ¹⁰⁵Ru,¹⁰⁷Hg. ²⁰³Hg, ^(121m)Te, ^(122m)Te, ^(125m)Te, ¹⁶⁵Tm, ¹⁶⁷Tm, ¹⁶⁸Tm,¹⁹⁷Pt, ¹⁰⁹Pd, ¹⁰⁵Pr, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au, ⁵⁷Co, ⁵⁸Co,⁵¹Cr, ⁵⁹Fe, ⁷⁵Se, ²⁰¹Tl, ²⁵⁵Ac. ⁷⁶Br, ¹⁶⁹Yb, and the like.

16. Radiosensitizers

The anti-B7-H3 antibodies of the invention may be conjugated to at leastone radiosensitizer. The term “radiosensitizer.” as used herein, isdefined as a molecule, preferably a low molecular weight molecule,administered to animals in therapeutically effective amounts to increasethe sensitivity of the cells to be radiosensitized to electromagneticradiation and/or to promote the treatment of diseases that are treatablewith electromagnetic radiation. Radiosensitizers are agents that makecancer cells more sensitive to radiation therapy, while typically havingmuch less of an effect on normal cells. Thus, the radiosensitizer can beused in combination with a radiolabeled antibody or ADC. The addition ofthe radiosensitizer can result in enhanced efficacy when compared totreatment with the radiolabeled antibody or antibody fragment alone.Radiosensitizers are described in D. M. Goldberg (ed.). Cancer Therapywith Radiolabeled Antibodies. CRC Press (1995). Examples ofradiosensitizers include gemcitabine, 5-fluorouracil, taxane, andcisplatin.

Radiosensitizers may be activated by the electromagnetic radiation ofX-rays. Representative examples of X-ray activated radiosensitizersinclude, but are not limited to, the following: metronidazole,misonidazole, desmethylmisonidazole, pimonidazole, etanidazole,nimorazole, mitomycin C, RSU 1069. SR 4233, E09, RB 6145, nicotinamide,5-bromodeoxyuridine (BUdR). 5-iododeoxyuridine (IUdR),bromodeoxycytidine, fluorodeoxyuridine (FUdR), hydroxyurea, cisplatin,and therapeutically effective analogs and derivatives of the same.Alternatively, radiosensitizers may be activated using photodynamictherapy (PDT). Representative examples of photodynamic radiosensitizersinclude, but are not limited to, hematoporphyrin derivatives.Photofrin(r), benzoporphyrin derivatives, NPe6, tin etioporphyrin(SnET2), pheoborbide a, bacteriochlorophyll a, naphthalocyanines,phthalocyanines, zinc phthalocyanine, and therapeutically effectiveanalogs and derivatives of the same.

16. Topoisomerase Inhibitors

The anti-B7-H3 antibodies of the invention may be conjugated to at leastone topoisomerase inhibitor. Topoisomerase inhibitors are chemotherapyagents designed to interfere with the action of topoisomerase enzymes(topoisomerase I and II), which are enzymes that control the changes inDNA structure by catalyzing then breaking and rejoining of thephosphodiester backbone of DNA strands during the normal cell cycle.Representative examples of DNA topoisomerase I inhibitors include, butare not limited to, camptothecins and its derivatives irinotecan(CPT-11, Camptosar, Pfizer. Inc.) and topotecan (Hycamtin,GlaxoSmithKline Pharmaceuticals). Representative examples of DNAtopoisomerase II inhibitors include, but are not limited to, amsacrine,daunorubicin, doxotrubicin, epipodophyllotoxins, ellipticines,epirubicin, etoposide, razoxane, and teniposide.

17. Knase Inhibitors

The anti-B7-H3 antibodies of the invention may be conjugated to at leastone kinase inhibitor. By blocking the ability of protein kinases tofunction, tumor growth may be inhibited. Examples of kinase inhibitorsthat may be used in the ADCs of the invention include, but are notlimited to, Axitinib, Bosutinib, Cediranib, Dasatinib, Erlotinib,Gefitinib, Imatinib, Lapatinib, Lestaurtinib, Nilotinib, Semaxanib.Sunitinib, Osimertinib. Cobimetinib, Trametinib, Dabrafenib. Dinaciclib,and Vandetanib.

18. Other Agents

Examples of other agents that may be used in the ADCs of the inventioninclude, but are not limited to, abrin (e.g, abrin A chain), alphatoxin, Aleurites fordii proteins, amatoxin, crotin, curcin, dianthinproteins, diptheria toxin (e.g. diphtheria A chain and nonbinding activefragments of diphtheria toxin), deoxyribonuclease (Dnase), gelonin,mitogellin, modeccin A chain, Momordica charantia inhibitor, neomycin,onconase, phenomycin. Phytolaca americana proteins (PAPI, PAPII, andPAP-S), pokeweed antiviral protein, Pseudomonas endotoxin, Pseudomonasexotoxin (e.g. exotoxin A chain (from Pseudomonas aeruginosa)),restrictocin, ricin A chain, ribonuclease (Rnase), Sapaonariaofficinalis inhibitor, saporin, alpha-sarcin. Staphylcoccalenterotoxin-A, tetanus toxin, cisplatin, carboplatin, and oxaliplatin(Eloxatin, Sanofi Aventis), proteasome inhibitors (e.g. PS-341[bortezomib or Velcade]), HDAC inhibitors (vorinostat (Zolinza. Merck &Company, Inc.)), belinostat, entinostat, mocetinostat, andpanobinostat). COX-2 inhibitors, substituted ureas, heat shock proteininhibitors (e.g. Geldanamycin and its numerous analogs), adrenocorticalsuppressants, and the tricothecenes. (See, for example WO 93/21232).Other agents also include asparaginase (Espar, Lundbeck Inc.),hydroxyurea, levamisole, mitotane (Lysodren, Bristol-Myers Squibb), andtretinoin (Renova, Valeant Pharmaceuticals Inc.).

III.C. Anti-B7-H3 ADCs: Other Exemplary Linkers

In addition to the linkers mentioned above, other exemplary linkersinclude, but are not limited to, 6-maleimidocaproyl, maleimidopropanoyl(“MP”), valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine(“ala-phe”), p-aminobenzyloxycarbonyl (a “PAB”), N-Succinimidyl4-(2-pyridylthio) pentanoate (“SPP”), and4-(N-maleimidomethyl)cyclohexane-1 carboxylate (“MCC”).

In one aspect, an anti-B7-H3 antibody is conjugated to a drug, (such asauristatin, e.g., MMAE), via a linker comprising maleimidocaproyl(“mc”), valine citrulline (val-cit or “vc”), and PABA (referred to as a“mc-vc-PABA linker”). Maleimidocaproyl acts as a linker to theanti-B7-H3 antibody and is not cleavable. Val-cit is a dipeptide that isan amino acid unit of the linker and allows for cleavage of the linkerby a protease, specifically the protease cathepsin B. Thus, the val-citcomponent of the linker provides a means for releasing the auristatinfrom the ADC upon exposure to the intracellular environment. Within thelinker, p-aminobenzylalcohol (PABA) acts as a spacer and is selfimmolative, allowing for the release of the MMAE. The structure of themc-vc-PABA-MMAE linker is provided in FIG. 3.

As described above, suitable linkers include, for example, cleavable andnon-cleavable linkers. A linker may be a “cleavable linker,”facilitating release of a drug. Nonlimiting exemplary cleavable linkersinclude acid-labile linkers (e.g., comprising hydrazone),protease-sensitive (e.g., peptidase-sensitive) linkers, photolabilelinkers, or disulfide-containing linkers (Chari et al., Cancer Research52:127-131 (1992); U.S. Pat. No. 5,208,020). A cleavable linker istypically susceptible to cleavage under intracellular conditions.Suitable cleavable linkers include, for example, a peptide linkercleavable by an intracellular protease, such as lysosomal protease or anendosomal protease. In exemplary embodiments, the linker can be adipeptide linker, such as a valine-citrulline (val-cit) or aphenylalanine-lysine (phe-lys) linker.

Linkers are preferably stable extracellularly in a sufficient manner tobe therapeutically effective. Before transport or delivery into a cell,the ADC is preferably stable and remains intact, i.e. the antibodyremains conjugated to the drug moiety. Linkers that are stable outsidethe target cell may be cleaved at some efficacious rate once inside thecell. Thus, an effective linker will: (i) maintain the specific bindingproperties of the antibody; (ii) allow delivery, e.g., intracellulardelivery, of the drug moiety; and (iii) maintain the therapeutic effect,e.g., cytotoxic effect, of a drug moiety.

In one embodiment, the linker is cleavable under intracellularconditions, such that cleavage of the linker sufficiently releases thedrug from the antibody in the intracellular environment to betherapeutically effective. In some embodiments, the cleavable linker ispH-sensitive, i.e., sensitive to hydrolysis at certain pH values.Typically, the pH-sensitive linker is hydrolyzable under acidicconditions. For example, an acid-labile linker that is hydrolyzable inthe lysosome (e.g., a hydrazone, semicarbazone, thiosemicarbazone,cis-aconitic amide, orthoester, acetal, ketal, or the like) can be used.(See, e.g., U.S. Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchikand Walker, 1999, Pharm. Therapeutics 83:67-123: Neville et al., 1989,Biol. Chem. 264:14653-14661.) Such linkers are relatively stable underneutral pH conditions, such as those in the blood, but are unstable atbelow pH 5.5 or 5.0, the approximate pH of the lysosome. In certainembodiments, the hydrolyzable linker is a thioether linker (such as,e.g., a thioether attached to the therapeutic agent via an acylhydrazonebond (see, e.g., U.S. Pat. No. 5,622,929).

In other embodiments, the linker is cleavable under rducing conditions(e.g., a disulfide linker). A variety of disulfide linkers are known inthe art, including, for example, those that can be formed using SATA(N-succinimidyl-5-acetylthioacetate), SPDP(N-succinimidyl-3-(2-pyridyldithio)propionate). SPDB(N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT(N-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene),SPDB and SMPT. (See, e.g., Thorpe et al., 1987. Cancer Res.47:5924-5931; Wawrzynczak et al., In Immunoconjugates: AntibodyConjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed.,Oxford U. Press, 1987. See also U.S. Pat. No. 4,880,935.).

In some embodiments, the linker is cleavable by a cleaving agent, e.g.,an enzyme, that is present in the intracellular environment (e.g.,within a lysosome or endosom or caveolea). The linker can be, e.g., apeptidyl linker that is cleaved by an intracellular peptidase orprotease enzyme, including, but not limited to, a lysosomal or endosomalprotease. In some embodiments, the peptidyl linker is at least two aminoacids long or at least three amino acids long. Cleaving agents caninclude cathepsins B and D and plasmin, all of which are known tohydrolyze dipeptide drug derivatives resulting in the release of activedrug inside target cells (see, e.g., Dubowchik and Walker, 1999. Pharm.Therapeutics 83:67-123). Most typical are peptidyl linkers that arecleavable by enzymes that are present in B7-H3-expressing cells.Examples of such linkers are described, e.g., in U.S. Pat. No.6,214,345, incorporated herein by reference in its entirety and for allpurposes. In a specific embodiment, the peptidyl linker cleavable by anintracellular protease is a Val-Cit linker or a Phe-Lys linker (see.e.g., U.S. Pat. No. 6,214,345, which describes the synthesis ofdoxorubicin with the val-cit linker). One advantage of usingintracellular proteolytic release of the therapeutic agent is that theagent is typically attenuated when conjugated and the serum stabilitiesof the conjugates are typically high.

In other embodiments, the linker is a malonate linker (Johnson et al.,1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau etal., 1995. Bioorg-Med-Chem. 3(10):1299-1304), or a 3′-N-amide analog(Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1305-12).

In yet other embodiments, the linker unit is not cleavable and the drugis released, for example, by antibody degradation. See U.S. PublicationNo. 20050238649 incorporated by reference herein in its entirety. An ADCcomprising a non-cleavable linker may be designed such that the ADCremains substantially outside the cell and interacts with certainreceptors on a target cell surface such that the binding of the ADCinitiates (or prevents) a particular cellular signaling pathway.

In some embodiments, the linker is substantially hydrophilic linker(e.g., PEG4Mal and sulfo-SPDB). A hydrophilic linker may be used toreduce the extent to which the drug may be pumped out of resistantcancer cells through MDR (multiple drug resistance) or functionallysimilar transporters.

In other embodiments, upon cleavage, the linker functions to directly orindirectly inhibit cell growth and/or cell proliferation. For example,in some embodiments, the linker, upon cleavage, can function as anintercalating agent, thereby inhibiting macromolecular biosynthesis(e.g. DNA replication, RNA transcription, and/or protein synthesis).

In other embodiments, the linker is designed to facilitate bystanderkilling (the killing of neighboring cells) through diffusion of thelinker-drug and/or the drug alone to neighboring cells. In other,embodiments, the linker promotes cellular internalization.

The presence of a sterically hindered disulfide can increase thestability of a particular disulfide bond, enhancing the potency of theADC. Thus, in one embodiment, the linker includes a sterically hindereddisulfide linkage. A sterically hindered disulfide refers to a disulfidebond present within a particular molecular environment, wherein theenvironment is characterized by a particular spatial arrangement ororientation of atoms, typically within the same molecule or compound,which prevents or at least partially inhibits the reduction of thedisulfide bond. Thus, the presence of bulky (or sterically hindering)chemical moieties and/or bulky amino acid side chains proximal to thedisulfide bond prevents or at least partially inhibits the disulfidebond from potential interactions that would result in the reduction ofthe disulfide bond.

Notably, the aforementioned linker types are not mutually exclusive. Forexample, in one embodiment, the linker used in the anti-B7-H3 ADCsdescribed herein is a non-cleavable linker that promotes cellularinternalization.

In some embodiments, a linker component comprises a “stretcher unit”that links an antibody to another linker component or to a drug moiety.An illustrative stretcher unit described in U.S. Pat. No. 8,309,093,incorporated by reference herein. In certain embodiments, the stretcherunit is linked to the anti-B7-H3 antibody via a disulfide bond between asulfur atom of the anti-B7-H3 antibody unit and a sulfur atom of thestretcher unit. A representative stretcher unit of this embodiment isdepicted in U.S. Pat. No. 8,309,093, incorporated by reference herein.In yet other embodiments, the stretcher contains a reactive site thatcan form a bond with a primary or secondary amino group of an antibody.

Examples of these reactive sites include but are not limited to,activated esters such as succinimide esters, 4 nitrophenyl esters,pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acidchlorides, sulfonyl chlorides, isocyanates and isothiocyanates.Representative stretcher units of this embodiment are depicted in U.S.Pat. No. 8,309,093, incorporated by reference herein.

In some embodiments, the stretcher contains a reactive site that isreactive to a modified carbohydrate's (—CHO) group that can be presenton an antibody. For example, a carbohydrate can be mildly oxidized usinga reagent such as sodium periodate and the resulting (—CHO) unit of theoxidized carbohydrate can be condensed with a Stretcher that contains afunctionality such as a hydrazide, an oxime, a primary or secondaryamine, a hydrazine, a thiosemicarbazone, a hydrazine carboxylate, and anarylhydrazide such as those described by Kaneko et al., 1991.Bioconjugate Chem. 2:133-41. Representative Stretcher units of thisembodiment are depicted in U.S. Pat. No. 8,309,093, incorporated byreference herein.

In some embodiments, a linker component comprises an “amino acid unit”.In some such embodiments, the amino acid unit allows for cleavage of thelinker by a protease, thereby facilitating release of the drug from theimmunoconjugate upon exposure to intracellular proteases, such aslysosomal enzymes (Doronina et al. (2003) Nat. Biotechnol. 21:778-784).Exemplary amino acid units include, but are not limited to, dipeptides,tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptidesinclude, but are not limited to, valine-citrulline (vc or val-cit),alanine-phenylalanine (af or ala-phe); phenylalanine-lysine (fk orphe-lys); phenylalanine-homolysine (phe-homolys); andN-methyl-valine-citrulline (Me-val-cit). Exemplary tripeptides include,but are not limited to, glycine-valine-citrulline (gly-val-cit) andglycine-glycine-glycine (gly-gly-gly). An amino acid unit may compriseamino acid residues that occur naturally and/or minor amino acids and/ornon-naturally occurring amino acid analogs, such as citrulline Aminoacid units can be designed and optimized for enzymatic cleavage by aparticular enzyme, for example, a tumor-associated protease, cathepsinB. C and D, or a plasmin protease.

In one embodiment, the amino acid unit is valine-citrulline (vc orval-cit). In another aspect, the amino acid unit is phenylalanine-lysine(i.e., fk). In yet another aspect of the amino acid unit, the amino acidunit is N-methylvaline-citrulline. In yet another aspect, the amino acidunit is 5-aminovaleric acid, homo phenylalanine lysine,tetraisoquinolincarboxylate lysine, cyclohexylalanine lysine,isonepecotic acid lysine, beta-alanine lysine, glycine serine valineglutamine and isonepecotic acid.

Alternatively, in some embodiments, the amino acid unit is replaced by aglucuronide unit that links a stretcher unit to a spacer unit if thestretcher and spacer units are present, links a stretcher unit to thedrug moiety if the spacer unit is absent, and links the linker unit tothe drug if the stretcher and spacer units are absent. The glucuronideunit includes a site that can be cleaved by a β-glucuronidase enzyme(See also US 2012/0107332, incorporated by reference herein). In someembodiments, the glucuronide unit comprises a sugar moiety (Su) linkedvia a glycoside bond (—O′—) to a self-immolative group (Z) of theformula as depicted below (See also US 2012/0107332, incorporated byreference herein).

Su-O′—Z

The glycosidic bond (—O′—) is typically a β-glucuronidase-cleavage site,such as a bond cleavable by human, lysosomal β-glucuronidase. In thecontext of a glucuronide unit, the term “self-immolative group” refersto a di- or tri-functional chemical moiety that is capable of covalentlylinking together two or three spaced chemical moieties (i.e., the sugarmoiety (via a glycosidic bond), a drug moiety (directly or indirectlyvia a spacer unit), and, in some embodiments, a linker (directly orindirectly via a stretcher unit) into a stable molecule. Theself-immolative group will spontaneously separate from the firstchemical moiety (e.g., the spacer or drug unit) if its bond to the sugarmoiety is cleaved.

In some embodiments, the sugar moiety (Su) is cyclic hexose, such as apyranose, or a cyclic pentose, such as a furanose. In some embodiments,the pyranose is a glucuronide or hexose. The sugar moiety is usually inthe β-D conformation. In a specific embodiment, the pyranose is ap-D-glucuronide moiety (i.e., β-D-glucuronic acid linked to theself-immolative group —Z— via a glycosidic bond that is cleavable byβ-glucuronidase). In some embodiments, the sugar moiety is unsubstituted(e.g., a naturally occurring cyclic hexose or cyclic pentose). In otherembodiments, the sugar moiety can be a substituted β-D-glucuronide(i.e., glucuronic acid substituted with one or more group, suchhydrogen, hydroxyl, halogen, sulfur, nitrogen or lower alkyl. In someembodiments, the glucuronide unit has one of the formulas as describedin US 2012/0107332, incorporated by reference herein.

In some embodiments, the linker comprises a spacer unit (—Y—), which,when present, links an amino acid unit (or Glucuronide unit, see also US2012/0107332, incorporated by reference herein) to the drug moiety whenan amino acid unit is present. Alternately, the spacer unit links thestretcher unit to the drug moiety when the amino acid unit is absent.The spacer unit may also links the drug unit to the antibody unit whenboth the amino acid unit and stretcher unit are absent.

Spacer units are of two general types: non self-immolative orself-immolative. A non self-immolative spacer unit is one in which partor all of the spacer unit remains bound to the drug moiety aftercleavage, particularly enzymatic, of an amino acid unit (or glucuronideunit) from the antibody-drug conjugate. Examples of a nonself-immolative spacer unit include, but are not limited to a(glycine-glycine) spacer unit and a glycine spacer unit (see U.S. Pat.No. 8,309,093, incorporated by reference herein)).Other examples ofself-immolative spacers include, but are not limited to, aromaticcompounds that are electronically similar to the PAB group such as2-aminoimidazol-5-methanol derivatives (Hay et al., 1999. Bioorg. Med.Chem. Lett. 9:2237) and ortho or para-aminobenzylacetals. Spacers can beused that undergo cyclization upon amide bond hydrolysis, such assubstituted and unsubstituted 4-aminobutyric acid amides (Rodrigues etal., 1995, Chemistry Biology 2:223), appropriately substitutedbicyclo[2.2.1] and bicyclo[2,2,2] ring systems (Storm et al., 1972, J.Amer. Chem. Soc. 94:5815) and 2-aminophenylpropionic acid amides(Amsberry et al., 1990. J. Org. Chem. 55:5867). Elimination ofamine-containing drugs that are substituted at the α-position of glycine(Kingsbury et al., 1984, J. Med. Chem. 27:1447) are also examples ofself-immolative spacers.

Other examples of self-immolative spacers include, but are not limitedto, aromatic compounds that are electronically similar to the PAB groupsuch as 2-aminoimidazol-5-methanol derivatives (see, e.g., Hay et al.,1999, Bioorg. Med. Chem. Lett. 9:2237) and ortho orpara-aminobenzylacetals. Spacers can be used that undergo cyclizationupon amide bond hydrolysis, such as substituted and unsubstituted4-aminobutyric acid amides (see, e.g., Rodrigues et al., 1995, ChemistryBiology 2:223), appropriately substituted bicyclo[2.2.1] andbicyclo[2,2,2] ring systems (see, e.g., Storm et al., 1972, J. Amer.Chem. Soc. 94:5815) and 2-aminophenylpropionic acid amides (see. e.g.,Amsberry et al., 1990, J. Org. Chem. 55:5867). Elimination ofamine-containing drugs that are substituted at the α-position of glycine(see, e.g., Kingsbury et al., 1984, J. Med. Chem. 27:1447) are alsoexamples of self-immolative spacers.

Other suitable spacer units are disclosed in Published U.S. PatentApplication No. 2005-0238649, the disclosure of which is incorporated byreference herein.

Another approach for the generation of ADCs involves the use ofheterobifunctional cross-linkers which link the anti-B7-H3 antibody tothe drug moiety. Examples of cross-linkers that may be used includeN-succinimidyl 4-(5-nitro-2-pyridyldithio)-pentanoate or the highlywater-soluble analog N-sulfosuccinimidyl4-(5-nitro-2-pyridyldithio)-pentanoate.N-succinimidyl-4-(2-pyridyldithio) butyrate (SPDB),N-succinimidyl-4-(5-nitro-2-pyridyldithio) butyrate (SNPB), andN-sulfosuccinimidyl-4-(5-nitro-2-pyridyldithio) butyrate (SSNPB),N-succinimidyl-4-methyl-4-(5-nitro-2-pyridyldithio)pentanoate (SMNP),N-succinimidyl-4-(5-N,N-dimethylcarboxamido-2-pyridyldithio) butyrate(SCPB) orN-sulfosuccinimidyl4-(5-N,N-dimethylcarboxamido-2-pyridyldithio)butyrate (SSCPB)). The antibodies of the invention may be modified withthe cross-linkers N-succinimidyl 4-(5-nitro-2-pyridyldithio)-pentanoate,N-sulfosuccinimidyl 4-(5-nitro-2-pyridyldithio)-pentanoate, SPDB, SNPB,SSNPB, SMNP, SCPB, or SSCPB can then react with a small excess of aparticular drug that contains a thiol moiety to give excellent yields ofan ADC. Preferably, the cross-linkers are compounds of the formula asdepicted in U.S. Pat. No. 6,913,748, incorporated by reference herein.

In one embodiment, charged linkers (also referred to as pro-chargedlinkers) are used to conjugate anti-B7-H3 antibodies to drugs to formADCs. Charged linkers include linkers that become charged after cellprocessing. The presence of a charged group(s) in the linker of aparticular ADC or on the drug after cellular processing provides severaladvantages, such as (i) greater water solubility of the ADC, (ii)ability to operate at a higher concentration in aqueous solutions, (iii)ability to link a greater number of drug molecules per antibody,potentially resulting in higher potency. (iv) potential for the chargedconjugate species to be retained inside the target cell, resulting inhigher potency, and (v) improved sensitivity of multidrug resistantcells, which would be unable to export the charged drug species from thecell. Examples of some suitable charged or pro-charged cross-linkers andtheir synthesis are shown in FIGS. 1 to 10 of U.S. Pat. No. 8,236,319,and are incorporated by reference herein. Preferably, the charged orpro-charged cross-linkers are those containing sulfonate, phosphate,carboxyl or quaternary amine substituents that significantly increasethe solubility of the ADCs, especially for ADCs with 2 to 20 conjugateddrugs. Conjugates prepared from linkers containing a pro-charged moietywould produce one or more charged moieties after the conjugate ismetabolized in a cell.

Additional examples of linkers that can be used with the compositionsand methods include valine-citrulline; maleimidocaproyl; amino benzoicacids; p-aminobenzylcarbamoyl (PAB); lysosomal enzyme-cleavable linkers;maleimidocaproyl-polyethylene glycol (MC(PEG)6-OH); N-methyl-valinecitrulline; N-succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC); N-Succinimidyl4-(2-pyridyldithio)butanoate (SPDB); and N-Succinimidyl4-(2-pyridylthio)pentanoate (SPP) (See also US 2011/0076232). Anotherlinker for use in the invention includes an avidin-biotin linkage toprovide an avidin-biotin-containing ADC (See also U.S. Pat. No.4,676,980, PCT publication Nos. WO992/022332A2, WO1994/016729A1,WO1995/015770A1. WO1997/031655A2, WO1998/035704A1, WO1999/019500A1,WO2001/09785A2, WO2001/090198A1, WO2003/093793A2, WO2004050016A2,WO2005/081898A2, WO2006/083562A2, WO2006/089668A, WO2007/150020A1.WO2008/135237A1, WO2010/111198A1. WO2011/057216A1, WO2011/058321A1,WO2012/027494A1, and EP77671 B1), wherein some such linkers areresistant to biotinidase cleavage. Additional linkers that may be usedin the invention include a cohesin/dockerin pair to provide acohesion-dockerin-containing ADC (See PCT publication Nos.WO2008/097866A2, WO2008/097870A2, WO2008/103947A2, and WO2008/103953A2).

Additional linkers for use in the invention may contain non-peptidepolymers (examples include, but are not limited to, polyethylene glycol,polypropylene glycol, polyoxyethylated polyols, polyvinyl alcohol,polysaccharides, dextran, polyvinyl ethyl ether, PLA (poly(lacticacid)), PLGA (poly(lactic acid-glycolic acid)), and combinationsthereof, wherein a preferred polymer is polyethylene glycol) (See alsoPCT publication No. WO2011/000370). Additional linkers are alsodescribed in WO 2004-010957, U.S. Publication No. 20060074008. U.S.Publication No. 20050238649, and U.S. Publication No. 20060024317, eachof which is incorporated by reference herein in its entirety).

For an ADC comprising a maytansinoid, many positions on maytansinoidscan serve as the position to chemically link the linking moiety. In oneembodiment, maytansinoids comprise a linking moiety that contains areactive chemical group are C-3 esters of maytansinol and its analogswhere the linking moiety contains a disulfide bond and the chemicalreactive group comprises a N-succinimidyl or N-sulfosuccinimidyl ester.For example, the C-3 position having a hydroxyl group, the C-14 positionmodified with hydroxymethyl, the C-15 position modified with hydroxy andthe C-20 position having a hydroxy group are all useful. The linkingmoiety most preferably is linked to the C-3 position of maytansinol.

The conjugation of the drug to the antibody via a linker can beaccomplished by any technique known in the art. A number of differentreactions are available for covalent attachment of drugs and linkers toantibodies. This may be accomplished by reaction of the amino acidresidues of the antibody, including the amine groups of lysine, the freecarboxylic acid groups of glutamic and aspartic acid, the sulfhydrylgroups of cysteine and the various moieties of the aromatic amino acids.One of the most commonly used non-specific methods of covalentattachment is the carbodiimide reaction to link a carboxy (or amino)group of a compound to amino (or carboxy) groups of the antibody.Additionally, bifunctional agents such as dialdehydes or imidoestershave been used to link the amino group of a compound to amino groups ofan antibody. Also available for attachment of drugs to antibodies is theSchiff base reaction. This method involves the periodate oxidation of adrug that contains glycol or hydroxy groups, thus forming an aldehydewhich is then reacted with the binding agent. Attachment occurs viaformation of a Schiff base with amino groups of the antibody.Isothiocyanates can also be used as coupling agents for covalentlyattaching drugs to antibodies. Other techniques are known to the skilledartisan and within the scope of the invention.

In certain embodiments, an intermediate, which is the precursor of thelinker, is reacted with the drug under appropriate conditions. Incertain embodiments, reactive groups are used on the drug or theintermediate. The product of the reaction between the drug and theintermediate, or the derivatized drug, is subsequently reacted with theanti-B7-H3 antibody under appropriate conditions. The synthesis andstructure of exemplary linkers, stretcher units, amino acid units,self-immolative spacer units are described in U.S. Patent ApplicationPublication Nos. 20030083263, 20050238649 and 20050009751, each if whichis incorporated herein by reference.

Stability of the ADC may be measured by standard analytical techniquessuch as mass spectroscopy, HPLC, and the separation/analysis techniqueLCMS.

IV. Purification of Anti-B7-H3 ADCs

Purification of the ADCs may be achieved in such a way that ADCs havingcertain DARs are collected. For example, HIC resin may be used toseparate high drug loaded ADCs from ADCs having optimal drug to antibodyratios (DARs), e.g, a DAR of 4 or less. In one embodiment, a hydrophobicresin is added to an ADC mixture such that undesired ADCs, i.e., higherdrug loaded ADCs, bind the resin and can be selectively removed from themixture. In certain embodiments, separation of the ADCs may be achievedby contacting an ADC mixture (e.g., a mixture comprising a drug loadedspecies of ADC of 4 or less and a drug loaded species of ADC of 6 ormore) with a hydrophobic resin, wherein the amount of resin issufficient to allow binding of the drug loaded species which is beingremoved from the ADC mixture. The resin and ADC mixture are mixedtogether, such that the ADC species being removed (e.g., a drug loadedspecies of 6 or more) binds to the resin and can be separated from theother ADC species in the ADC mixture. The amount of resin used in themethod is based on a weight ratio between the species to be removed andthe resin, where the amount of resin used does not allow for significantbinding of the drug loaded species that is desired. Thus, methods may beused to reduce the average DAR to less than 4. Further, the purificationmethods described herein may be used to isolate ADCs having any desiredrange of drug loaded species, e.g., a drug loaded species of 4 or less,a drug loaded species of 3 or less, a drug loaded species of 2 or less,a drug loaded species of 1 or less.

Certain species of molecule(s) binds to a surface based on hydrophobicinteractions between the species and a hydrophobic resin. In oneembodiment, method of the invention refers to a purification processthat relies upon the intermixing of a hydrophobic resin and a mixture ofADCs, wherein the amount of resin added to the mixture determines whichspecies (e.g., ADCs with a DAR of 6 or more) will bind. Followingproduction and purification of an antibody from an expression system(e.g., a mammalian expression system), the antibody is reduced andcoupled to a drug through a conjugation reaction. The resulting ADCmixture often contains ADCs having a range of DARs, e.g., 1 to 8. In oneembodiment, the ADC mixture comprises a drug loaded species of 4 or lessand a drug loaded species of 6 or more. According to the methods of theinvention, the ADC mixture may be purified using a process, such as, butnot limited to, a batch process, such that ADCs having a drug loadedspecies of 4 or less are selected and separated from ADCs having ahigher drug load (e.g., ADCs having a drug loaded species of 6 or more).Notably, the purification methods described herein may be used toisolate ADCs having any desired range of DAR, e.g., a DAR of 4 or less,a DAR of 3 or less, or a DAR of 2 or less.

Thus, in one embodiment, an ADC mixture comprising a drug loaded speciesof 4 or less and a drug loaded species of 6 or more may be contactedwith a hydrophobic resin to form a resin mixture, wherein the amount ofhydrophobic resin contacted with the ADC mixture is sufficient to allowbinding of the drug loaded species of 6 or more to the resin but doesnot allow significant binding of the drug load species of 4 or less; andremoving the hydrophobic resin from the ADC mixture, such that thecomposition comprising ADCs is obtained, wherein the compositioncomprises less than 15% of the drug loaded species of 6 or more, andwherein the ADC comprises an antibody conjugated to a Bcl-xL inhibitor.In a separate embodiment, the method of the invention comprisescontacting an ADC mixture comprising a drug loaded species of 4 or lessand a drug loaded species of 6 or more with a hydrophobic resin to forma resin mixture, wherein the amount of hydrophobic resin contacted withthe ADC mixture is sufficient to allow binding of the drug loadedspecies of 6 or more to the resin but does not allow significant bindingof the drug load species of 4 or less: and removing the hydrophobicresin from the ADC mixture, such that the composition comprising ADCs isobtained, wherein the composition comprises less than 15% of the drugloaded species of 6 or more, and wherein the ADC comprises an antibodyconjugated to a Bcl-xL inhibitor, wherein the hydrophobic resin weightis 3 to 12 times the weight of the drug loaded species of 6 or more inthe ADC mixture.

The ADC separation method described herein method may be performed usinga batch purification method. The batch purification process generallyincludes adding the ADC mixture to the hydrophobic resin in a vessel,mixing, and subsequently separating the resin from the supernatant. Forexample, in the context of batch purification, a hydrophobic resin maybe prepared in or equilibrated to the desired equilibration buffer. Aslurry of the hydrophobic resin may thus be obtained. The ADC mixturemay then be contacted with the slurry to adsorb the specific species ofADC(s) to be separated by the hydrophobic resin. The solution comprisingthe desired ADCs that do not bind to the hydrophobic resin material maythen be separated from the slurry. e.g., by filtration or by allowingthe slurry to settle and removing the supernatant. The resulting slurrycan be subjected to one or more washing steps. In order to elute boundADCs, the salt concentration can be decreased. In one embodiment, theprocess used in the invention includes no more than 50 g of hydrophobicresin.

Thus, a batch method may be used to contact an ADC mixture comprising adrug loaded species of 4 or less and a drug loaded species of 6 or morewith a hydrophobic resin to form a resin mixture, wherein the amount ofhydrophobic resin contacted with the ADC mixture is sufficient to allowbinding of the drug loaded species of 6 or more to the resin but doesnot allow significant binding of the drug load species of 4 or less; andremoving the hydrophobic resin from the ADC mixture, such that thecomposition comprising ADCs is obtained, wherein the compositioncomprises less than 15% of the drug loaded species of 6 or more, andwherein the ADC comprises an antibody conjugated to a Bcl-xL inhibitor.In a separate embodiment, a batch method is used to contact an ADCmixture comprising a drug loaded species of 4 or less and a drug loadedspecies of 6 or more with a hydrophobic resin to form a resin mixture,wherein the amount of hydrophobic resin contacted with the ADC mixtureis sufficient to allow binding of the drug loaded species of 6 or moreto the resin but does not allow significant binding of the drug loadspecies of 4 or less; and removing the hydrophobic resin from the ADCmixture, such that the composition comprising ADCs is obtained, whereinthe composition comprises less than 15% of the drug loaded species of 6or more, and wherein the ADC comprises an antibody conjugated to aBcl-xL inhibitor, wherein the hydrophobic resin weight is 3 to 12 timesthe weight of the drug loaded species of 6 or more in the ADC mixture.

Alternatively, in a separate embodiment, purification may be performedusing a circulation process, whereby the resin is packed in a containerand the ADC mixture is passed over the hydrophobic resin bed until thespecific species of ADC(s) to be separated have been removed. Thesupernatant (containing the desired ADC species) is then pumped from thecontainer and the resin bed may be subjected to washing steps.

A circulation process may be used to contact an ADC mixture comprising adrug loaded species of 4 or less and a drug loaded species of 6 or morewith a hydrophobic resin to form a resin mixture, wherein the amount ofhydrophobic resin contacted with the ADC mixture is sufficient to allowbinding of the drug loaded species of 6 or more to the resin but doesnot allow significant binding of the drug load species of 4 or less; andremoving the hydrophobic resin from the ADC mixture, such that thecomposition comprising ADCs is obtained, wherein the compositioncomprises less than 15% of the drug loaded species of 6 or more, andwherein the ADC comprises an antibody conjugated to a Bcl-xL inhibitor.In a separate embodiment, a circulation process is used to contact anADC mixture comprising a drug loaded species of 4 or less and a drugloaded species of 6 or more with a hydrophobic resin to form a resinmixture, wherein the amount of hydrophobic resin contacted with the ADCmixture is sufficient to allow binding of the drug loaded species of 6or more to the resin but does not allow significant binding of the drugload species of 4 or less; and removing the hydrophobic resin from theADC mixture, such that the composition comprising ADCs is obtained,wherein the composition comprises less than 15% of the drug loadedspecies of 6 or more, and wherein the ADC comprises an antibodyconjugated to a Bcl-xL inhibitor, wherein the hydrophobic resin weightis 3 to 12 times the weight of the drug loaded species of 6 or more inthe ADC mixture.

Alternatively, a flow through process may be used to purify an ADCmixture to arrive at a composition comprising a majority of ADCs havinga certain desired DAR. In a flow through process, resin is packed in acontainer, e.g., a column, and the ADC mixture is passed over the packedresin such that the desired ADC species does not substantially bind tothe resin and flows through the resin, and the undesired ADC species isbound to the resin. A flow through process may be performed in a singlepass mode (where the ADC species of interest are obtained as a result ofa single pass through the resin of the container) or in a multi-passmode (where the ADC species of interest are obtained as a result ofmultiple passes through the resin of the container). The flow throughprocess is performed such that the weight of resin selected binds to theundesired ADC population, and the desired ADCs (e.g., DAR 2-4) flow overthe resin and are collected in the flow through after one or multiplepasses.

A flow through process may be used to contact an ADC mixture comprisinga drug loaded species of 4 or less and a drug loaded species of 6 ormore with a hydrophobic resin, wherein the amount of hydrophobic resincontacted with the ADC mixture is sufficient to allow binding of thedrug loaded species of 6 or more to the resin but does not allowsignificant binding of the drug load species of 4 or less, where thedrug load species of 4 or less passes over the resin and is subsequentlycollected after one or multiple passes, such that the compositioncomprising the desired ADCs (e.g. DAR 2-4) is obtained, wherein thecomposition comprises less than 15% of the drug loaded species of 6 ormore, and wherein the ADC comprises an antibody conjugated to a Bcl-xLinhibitor. In a separate embodiment, a flow through process is used tocontact an ADC mixture comprising a drug loaded species of 4 or less anda drug loaded species of 6 or more with a hydrophobic resin by passingthe ADC mixture over the resin, wherein the amount of hydrophobic resincontacted with the ADC mixture is sufficient to allow binding of thedrug loaded species of 6 or more to the resin but does not allowsignificant binding of the drug load species of 4 or less, where thedrug load species of 4 or less passes over the resin and is subsequentlycollected, such that the composition comprising ADCs is obtained,wherein the composition comprises less than 15% of the drug loadedspecies of 6 or more, and wherein the ADC comprises an antibodyconjugated to an a drug, e.g., a Bcl-xL inhibitor, wherein the amount ofhydrophobic resin weight is 3 to 12 times the weight of the drug loadedspecies of 6 or more in the ADC mixture.

Following a flow through process, the resin may be washed with a one ormore washes following in order to further recover ADCs having thedesired DAR range (found in the wash filtrate). For example, a pluralityof washes having decreasing conductivity may be used to further recoverADCs having the DAR of interest. The elution material obtained from thewashing of the resin may be subsequently combined with the filtrateresulting from the flow through process for improved recovery of ADCshaving the DAR of interest.

The aforementioned batch, circulation, and flow through processpurification methods are based on the use of a hydrophobic resin toseparate high vs. low drug loaded species of ADC. Hydrophobic resincomprises hydrophobic groups which interact with the hydrophobicproperties of the ADCs. Hydrophobic groups on the ADC interact withhydrophobic groups within the hydrophobic resin. The more hydrophobic aprotein is the stronger it will interact with the hydrophobic resin.

Hydrophobic resin normally comprises a base matrix (e.g., cross-linkedagarose or synthetic copolymer material) to which hydrophobic ligands(e.g., alkyl or aryl groups) are coupled. Many hydrophobic resins areavailable commercially. Examples include, but are not limited to, PhenylSepharose™ 6 Fast Flow with low or high substitution (Pharmacia LKBBiotechnology, AB, Sweden); Phenyl Sepharos™ High Performance (PharmaciaLKB Biotechnology, AB, Sweden): Octyl Sepharose™ High Performance(Pharmacia LKB Biotechnology. AB. Sweden): Fractogel™ EMD Propyl orFractogel™ EMD Phenyl columns (E. Merck, Germany); Macro-Prep™ Methyl orMacro-Prep™, t-Butyl Supports (Bio-Rad, California): WP HI-Propyl (C₃)™(J. T. Baker, New Jersey); and Toyopearl™ ether, hexyl, phenyl or butyl(TosoHaas, PA). In one embodiment, the hydrophobic resin is a butylhydrophobic resin. In another embodiment, the hydrophobic resin is aphenyl hydrophobic resin. In another embodiment, the hydrophobic resinis a hexyl hydrophobic resin, an octyl hydrophobic resin, or a decylhydrophobic resin. In one embodiment, the hydrophobic resin is amethacrylic polymer having n-buty ligands (e.g. TOYOPEARL® Butyl-600M).

Further methods for purifying ADC mixtures to obtain a compositionhaving a desired DAR are described in U.S. application Ser. No.14/210,602 (U.S. Patent Appln. Publication No. US 20141028698),incorporated by reference in its entirety.

In certain embodiments of the invention, ADCs described herein having aDAR2 are purified from ADCs having higher or lower DARs. Such purifiedDAR2 ADCs are referred to herein as “E2”. Purification methods forachieving a composition having E2 anti-B7-H3 ADCs. In one embodiment, ofthe invention provides a composition comprising an ADC mixture, whereinat least 75% of the ADCs are anti-B7H3 ADCs (like those describedherein) having a DAR2. In another embodiment, the invention provides acomposition comprising an ADC mixture, wherein at least 80% of the ADCsare anti-B7H3 ADCs (like those described herein) having a DAR2. Inanother embodiment, the invention provides a composition comprising anADC mixture, wherein at least 85% of the ADCs are anti-B7H3 ADCs (likethose described herein) having a DAR2. In another embodiment, theinvention provides a composition comprising an ADC mixture, wherein atleast 90% of the ADCs are anti-B7H3 ADCs (like those described herein)having a DAR2.

V. Uses of Anti-B7-H3 Antibodies and Anti-B7-H3 ADCs

The antibodies and ADCs of the invention preferably are capable ofneutralizing human B7-H3 activity both in vivo and in vitro.Accordingly, such antibodies and ADCs of the invention can be used toinhibit hB7-H3 activity, e.g., in a cell culture containing hB7-H3, inhuman subjects or in other mammalian subjects having B7-H3 with which anantibody of the invention cross-reacts. In one embodiment, the inventionprovides a method for inhibiting hB7-H3 activity comprising contactinghB7-H3 with an antibody or ADC of the invention such that hB7-H3activity is inhibited. For example, in a cell culture containing, orsuspected of containing hB7-H3, an antibody or antibody portion of theinvention can be added to the culture medium to inhibit hB7-H3 activityin the culture.

In another embodiment, of the invention a method for reducing hB7-H3activity in a subject, advantageously from a subject suffering from adisease or disorder in which B7-H3 activity is detrimental. Theinvention provides methods for reducing B7-H3 activity in a subjectsuffering from such a disease or disorder, which method comprisesadministering to the subject an antibody or ADC of the invention suchthat B7-H3 activity in the subject is reduced. Preferably, the B7-H3 ishuman B7-H3, and the subject is a human subject. Alternatively, thesubject can be a mammal expressing a B7-H3 to which antibodies of theinvention are capable of binding. Still further the subject can be amammal into which B7-H3 has been introduced (e.g., by administration ofB7-H3 or by expression of a B7-H3 transgene). Antibodies or ADCs of theinvention can be administered to a human subject for therapeuticpurposes. Moreover, antibodies or ADCS of the invention can beadministered to a non-human mammal expressing a B7-H3 with which theantibody is capable of binding for veterinary purposes or as an animalmodel of human disease. Regarding the latter, such animal models may beuseful for evaluating the therapeutic efficacy of antibodies of theinvention (e.g., testing of dosages and time courses of administration).

As used herein, the term “a disorder in which B7-H3 expression isdetrimental” is intended to include diseases and other disorders inwhich the presence of B7-H3 in a subject suffering from the disorder hasbeen shown to be expressed, or has been shown to be or is suspected ofbeing either responsible for the pathophysiology of the disorder or afactor that contributes to the disorder. For example, the ADCs of theinvention may be used to target tumor cells that are expressing B7-H3.Non-limiting examples of disorders that can be treated with the ADCs ofthe invention, for example, an ADC comprising huAbl3v1, include, but arenot limited to, a variety of cancers including, but not limited to,small cell lung cancer, non small cell lunch cancer (NSCLC), breastcancer, ovarian cancer, lung cancer, a glioma, prostate cancer,pancreatic cancer, colon cancer, head and neck cancer, leukemia, e.g.,acute myeloid leukemia (AML), lymphoma, e.g., non-Hodgkin's lymphoma(NHL), and kidney cancer. Other examples of cancer that may be treatedusing the compositions and methods disclosed herein include squamouscell carcinoma (e.g., squamous lung cancer or squamous head and neckcancer), triple negative breast cancer, non-small cell lung cancer,colorectal cancer, and mesothelioma. In one embodiment, the antibodiesor ADCs disclosed herein are used to treat a solid tumor, e.g., inhibitgrowth of or decrease size of a solid tumor, overexpressing B7-H3 orwhich is B7-H3 positive. In one embodiment, the invention is directed tothe treatment of squamous lung cancer associated with B7-H3 expression.In another embodiment, the antibodies and ADCs disclosed herein are usedto treat triple negative breast cancer (TNBC). Diseases and disordersdescribed herein may be treated by anti-B7-H3 antibodies or ADCs of theinvention, as well as pharmaceutical compositions comprising suchanti-B7-H3 antibodies or ADCs.

In certain embodiments, the cancer may be characterized as having EGFRoverexpression. In one embodiment, the ADCs of the invention may be usedto treating cancer associated with an activating EGFR mutation. Examplesof such mutations include, but are not limited to, an exon 19 deletionmutation, a single-point substitution mutation L858R in exon 21, a T790Mpoint mutation, and combinations thereof.

In certain embodiments, the antibodies or ADCs disclosed herein areadministered to a subject in need thereof in order to treat advancedsolid tumor types likely to exhibit elevated levels of B7-H3. Examplesof such tumors include, but are not limited to, small cell lung cancer,breast cancer, ovarian cancer, head and neck squamous cell carcinoma,non-small cell lung cancer, triple negative breast cancer, colorectalcarcinoma, and glioblastoma multiforme.

In certain embodiments, the invention includes a method for inhibitingor decreasing solid tumor growth in a subject having a solid tumor, saidmethod comprising administering an anti-B7-H3 antibody or ADC describedherein, to the subject having the solid tumor, such that the solid tumorgrowth is inhibited or decreased. In certain embodiments, the solidtumor is a non-small cell lung carcinoma or a glioblastoma. In furtherembodiments, the solid tumor is a B7-H3-expressing solid tumors. Infurther embodiments, the solid tumor is an B7-H3 overexpressing solidtumors. In certain embodiments the anti-B7-H3 antibodies or ADCsdescribed herein are administered to a subject having glioblastomamultiforme, alone or in combination with an additional agent, e.g.,radiation and/or temozolomide.

In certain embodiments the anti-B7-H3 ADCs described herein are areadministered to a subject having small cell lung cancer, alone or incombination with an additional agent, e.g., ABT-199 (venetoclax).

In certain embodiments the anti-B7-H3 ADCs described herein areadministered to a subject having non-small cell lung cancer, alone or incombination with an additional agent, e.g., a taxane. In certainembodiments the anti-B7-H3 antibodies or ADCs described herein areadministered to a subject having breast cancer, alone or in combinationwith an additional agent, e.g., a taxane. In certain embodiments theanti-B7-H3 antibodies or ADCs described herein are administered to asubject having ovarian cancer, alone or in combination with anadditional agent, e.g., a taxane.

Other combination therapies which are included in the invention are theadministration of an anti-B7-H3 ADC with an agent selected from thegroup consisting of an anti-PD1 antibody (e.g. pembrolizumab), ananti-PD-L1 antibody (e.g., atezolizumab), an anti-CTLA-4 antibody (e.g.ipilimumab), a MEK inhibitor (e.g. trametinib), an ERK inhibitor, a BRAFinhibitor (e.g. dabrafenib), osimertinib, erlotinib, gefitinib,sorafenib, a CDK9 inhibitor (e.g. dinaciclib), a MCL-1 inhibitor,temozolomide, a Bcl-xL inhibitor, a Bcl-2 inhibitor (e.g. venetoclax),ibrutinib, a mTOR inhibitor (e.g. everolimus), a PI3K inhibitor (e.g.buparlisib), duvelisib, idelalisib, an AKT inhibitor, a HER2 inhibitor(e.g. lapatinib), a taxane (e.g., docetaxel, paclitaxel,nab-paclitaxel), venetoclax, an ADC comprising an auristatin, an ADCcomprising a PBD (e.g. rovalpituzumab tesirine), an ADC comprising amaytansinoid (e.g. TDM1), a TRAIL agonist, a proteasome inhibitor (e.g.bortezomib), and a nicotinamide phosphoribosyltransferase (NAMPT)inhibitor.

Combination therapies include administration of an ADC of the inventionprior to, concurrently with, or following administration of anadditional therapeutic agent, including those described above.

In certain embodiments, the invention includes a method for inhibitingor decreasing solid tumor growth in a subject having a solid tumor whichwas identified as an B7-H3 expressing or B7-H3 overexpressing tumor,said method comprising administering an anti-B7-H3 antibody or ADCdescribed herein, to the subject having the solid tumor, such that thesolid tumor growth is inhibited or decreased. Methods for identifyingB7-H3 expressing tumors (e.g., B7-H3 overexpressing tumors) are known inthe art, and include FDA-approved tests and validation assays. Forexample, the B7-H3 assay is a qualitative immunohistochemical (IHC) kitsystem used to identify B7-H3 expression in normal and neoplastictissues routinely-fixed for histological evaluation. In addition.PCR-based assays may also be used for identifying B7-H3 overexpressingtumors. The amplified PCR products may be subsequently analyzed, forexample, by gel electrophoresis using standard methods known in the artto determine the size of the PCR products. Such tests may be used toidentify tumors that may be treated with the methods and compositionsdescribed herein.

Any of the methods for gene therapy available in the art can be usedaccording to the invention. For general reviews of the methods of genetherapy, see Goldspiel et al., 1993, Clincal Pharmacy 12:488-505; Wu andWu. 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol.Toxicol. 32:573-596; Mulligan, Science 260:926-932 (1993); and Morganand Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May 1993, TIBTECH11(5):155-215. Methods commonly known in the art of recombinant DNAtechnology which can be used are described in Ausubel et al. (eds.),Current Protocols in Molecular Biology, John Wiley & Sons, N Y (1993);and Kriegler, Gene Transfer and Expression. A Laboratory Manual,Stockton Press, NY (190). Detailed description of various methods ofgene therapy is provided in US20050042664 A1 which is incorporatedherein by reference.

In another aspect, this application features a method of treating (e.g.,curing, suppressing, ameliorating, delaying or preventing the onset of,or preventing recurrence or relapse of) or preventing a B7-H3-associateddisorder, in a subject. The method includes: administering to thesubject an B7-H3 binding agent. e.g., an anti-B7-H3 antibody or fragmentthereof as described herein, in an amount sufficient to treat or preventthe B7-H3-associated disorder. The B7-H3 antagonist, e.g., theanti-B7-H3 antibody or fragment thereof, can be administered to thesubject, alone or in combination with other therapeutic modalities asdescribed herein.

Antibodies or ADCs of the invention, or antigen binding portions thereofcan be used alone or in combination to treat such diseases. It should beunderstood that the antibodies of the invention or antigen bindingportion thereof can be used alone or in combination with an additionalagent, e.g., a therapeutic agent, said additional agent being selectedby the skilled artisan for its intended purpose. For example, theadditional agent can be a therapeutic agent art-recognized as beinguseful to treat the disease or condition being treated by the antibodyof the invention. The additional agent also can be an agent that impartsa beneficial attribute to the therapeutic composition, e.g., an agentwhich affects the viscosity of the composition.

It should further be understood that the combinations which are to beincluded within this invention are those combinations useful for theirintended purpose. The agents set forth below are illustrative forpurposes and not intended to be limited. The combinations, which arepart of this invention, can be the antibodies of the invention and atleast one additional agent selected from the lists below. Thecombination can also include more than one additional agent, e.g., twoor three additional agents if the combination is such that the formedcomposition can perform its intended function.

The combination therapy can include one or more B7-H3 antagonists, e.g.,anti-B7-H3 antibodies or fragments thereof, formulated with, and/orco-administered with, one or more additional therapeutic agents, e.g.,one or more cytokine and growth factor inhibitors, immunosuppressants,anti-inflammatory agents (e.g., systemic anti-inflammatory agents),anti-fibrotic agents, metabolic inhibitors, enzyme inhibitors, and/orcytotoxic or cytostatic agents, mitotic inhibitors, antitumorantibiotics, immunomodulating agents, vectors for gene therapy,alkylating agents, antiangiogenic agents, antimetabolites,boron-containing agents, chemoprotective agents, hormones, antihormoneagents, corticosteroids, photoactive therapeutic agents,oligonucleotides, radionuclide agents, topoisomerase inhibitors, kinaseinhibitors, or radiosensitizers, as described in more herein.

In a particular embodiment, the anti-B7-H3 binding proteins describedherein, for example, anti-B7-H3 antibodies, are used in combination withan anti-cancer agent or an antineoplastic agent. The terms “anti-canceragent” and “antineoplastic agent” refer to drugs used to treatmalignancies, such as cancerous growths. Drug therapy may be used alone,or in combination with other treatments such as surgery or radiationtherapy. Several classes of drugs may be used in cancer treatment,depending on the nature of the organ involved. For example, breastcancers are commonly stimulated by estrogens, and may be treated withdrugs which inactive the sex hormones. Similarly, prostate cancer may betreated with drugs that inactivate androgens, the male sex hormone.Anti-cancer agents that may be used in conjunction with the anti-B7-H3antibodies or ADCs of the invention include, among others, an anti-PD1antibody (e.g., pembrolizumab), an anti-PD-L1 antibody (e.g.,atezolizumab), an anti-CTLA-4 antibody (e.g., ipilimumab), a MEKinhibitor (e.g., trametinib), an ERK inhibitor, a BRAF inhibitor (e.g.,dabrafenib), osimertinib (AZD9291), erlotinib, gefitinib, sorafenib, aCDK9 inhibitor (e.g., dinaciclib), a MCL-1 inhibitor, temozolomide, aBcl-xL inhibitor, a Bcl-2 inhibitor (e.g., venetoclax), ibrutinib, amTOR inhibitor (e.g., everolimus), a PI3K inhibitor (e.g., buparlisib),duvelisib, idelalisib, an AKT inhibitor, a HER2 inhibitor (e.g.,lapatinib), Herceptin, a taxane (e.g., docetaxel, paclitaxel,nab-paclitaxel), an ADC comprising an auristatin, an ADC comprising aPBD (e.g., rovalpituzumab tesirine), an ADC comprising a maytansinoid(e.g., TDM1), a TRAIL agonist, a proteasome inhibitor (e.g.,bortezomib), and a nicotinamide phosphoribosyltransferase (NAMPT)inhibitor, as well as the following agents:

Anti-Cancer Agent Comments Examples Antibodies Antibodies which bindIGF- A12 (fully humanized mAb) 1R (insulin-like growth 19D12 (fullyhumanized mAb) factor type 1 receptor), Cp751-871 (fully humanized mAb)which is expressed on the H7C10 (humanized mAb) cell surface of mosthuman alphaIR3 (mouse) cancers ScFV/FC (mouse/human chimera) EM/164(mouse) Antibodies which bind Matuzumab (EMD72000) EGFR; Mutationsaffecting Erbitux ®/Cetuximab (Imclone) EGFR expression or activityVectibix ®/Panitumumab (Amgen) could result in cancer mAb 806Nimotuxumab (TheraCIM) Antibodies which bind AVEO (AV299) (AVEO) cMET(Mesechymal AMG102 (Amgen) epithelial transition factor); 5D5 (OA-5d5)(Genentech) a member of the MET H244G11 (Pierre Fabre) family ofreceptor tyrosine kinases) Anti-ErbB3 antibodies Ab #14 (MM 121-14)Herceptin ® (Trastuzumab; Genentech) 1B4C3; 2D1D12 (U3 Pharma AG) SmallMolecules Insulin-like growth factor NVP-AEW541-A Targeting IGF1R type 1receptor which is BMS-536,924 (1H-benzoimidazol-2-yl)-1H- expressed onthe cell pyridin-2-one) surface of many human BMS-554,417 cancersCycloligan TAE226 PQ401 Small Molecules EGFR; Iressa ®/Gefitinib(AstraZeneca) Targeting EGFR Overexpression or CI-1033 (PD 183805)(Pfizer) mutations affecting EGFR Lapatinib (GW-572016)(GlaxoSmithKline) expression or activity could Tykerb ®/LapatinibDitosylate (Smith Kline result in cancer Beecham) Tarceva ®/ErlotinibHCL (OSI-774) (OSI Pharma) PKI-166 (Novartis) PD-158780 EKB-569Tyrphostin AG 1478 (4-(3-Chloroanillino)- 6,7-dimethoxyquinazoline)Small Molecules cMET (Mesenchymal PHA665752 Targeting cMET epithelialtransition factor); ARQ 197 a member of the MET family of receptortyrosine kinases) Antimetabolites Flourouracil (5-FU)Capecitabine/XELODA ® (HLR Roche) 5-Trifluoromethyl-2′-deoxyuridineMethotrexate sodium (Trexall) (Barr) Raltitrexed/Tomudex ® (AstraZeneca)Pemetrexed/Alimta ® (Lilly) Tegafur Cytosine Arabinoside (Cytarabine,Ara-C)/ Thioguanine ® (GlaxoSmithKline) 5-azacytidine 6-mercaptopurine(Mercaptopurine, 6-MP) Azathioprine/Azasan ® (AAIPHARMA LLC)6-thioguanine (6-TG)/Purinethol ® (TEVA) Pentostatin/Nipent ® (HospiraInc.) Fludarabine phosphate/Fludara ® (Bayer Health Care) Cladribine(2-CdA, 2-chlorodeoxyadenosine)/ Leustatin ® (Ortho Biotech) Alkylatingagents An alkylating antineoplastic Ribonucleotide Reductase Inhibitor(RNR) agent is an alkylating agent Cyclophosphamide/Cytoxan (BMS) thatattaches an alkyl group Neosar (TEVA) to DNA. Since cancer cellsIfosfamide/Mitoxana ® (ASTA Medica) generally proliferate Thiotepa(Bedford, Abraxis, Teva) unrestrictively more than do BCNU→1,3-bis(2-chloroethyl)-1-nitosourea healthy cells they are more CCNU→ 1,-(2-chloroethyl)-3-cyclohexyl-1- sensitive to DNA damage, nitrosourea(methyl CCNU) and alkylating agents are Hexamethylmelamine (Altretamine,HMM)/ used clinically to treat a Hexalen ® (MGI Pharma Inc.) variety oftumors. Busulfan/Myleran (GlaxoSmithKline) Procarbazine HCL/Matulane(Sigma Tau Pharmaceuticals, Inc.) Dacarbazine (DTIC)Chlorambucil/Leukara ® (SmithKline Beecham) Melphalan/Alkeran ®(GlaxoSmithKline) Cisplatin (Cisplatinum, CDDP)/Platinol (Bristol Myers)Carboplatin/Paraplatin (BMS) Oxaliplatin/Eloxitan ® (Sanofi-Aventis US)Topoisomerase Topoisomerase inhibitors Doxorubicin HCL/Doxil ® (Alza)inhibitors are chemotherapy agents Daunorubicin citrate/Daunoxome ®(Gilead) designed to interfere with Mitoxantrone HCL/Novantrone (EMD theaction of topoisomerase Serono) enzymes (topoisomerase I Actinomycin Dand II), which are enzymes Etoposide/Vepesid ® (BMS)/Etopophos ® thatcontrol the changes in (Hospira, Bedford, Teva Parenteral, Etc.) DNAstructure by Topotecan HCL/Hycamtin ® catalyzing the breaking and(GlaxoSmithKline) rejoining of the Teniposide (VM-26)/Vumon ® (BMS)phosphodiester backbone of Irinotecan HCL(CPT-ll/Camptosar ® DNA strandsduring the (Pharmacia & Upjohn) normal cell cycle. MicrotubuleMicrotubules are one of the Vincristine/Oncovin ® (Lilly) targetingagents components of the Vinblastine sulfate/Velban ®(discontinued)cytoskeleton. They have (Lilly) diameter of ~24 nm and Vinorelbinetartrate/Navelbine ® length varying from several (PierreFabre)micrometers to possibly Vindesine sulphate/Eldisine ® (Lilly)millimeters in axons of Paclitaxel/Taxol ® (BMS) nerve cells.Microtubules Docetaxel/Taxotere ® (Sanofi Aventis US) serve asstructural Nanoparticle paclitaxel (ABI-007)/ components within cellsand Abraxane ® (Abraxis BioScience, Inc.) are involved in manyIxabepilone/IXEMPRA ™ (BMS) cellular processes including mitosis,cytokinesis, and vesicular transport. Kinase inhibitors Kinases areenzymes that Imatinib mesylate/Gleevec (Novartis) catalyze the transferof Sunitinib malate/Sutent ® (Pfizer) phosphate groups from Sorafenibtosylate/Nexavar ® (Bayer) high-energy, phosphate- Nilotinibhydrochloride monohydrate/ donating molecules to Tasigna ® (Novartis),Osimertinib, specific substrates, and are Cobimetinib, Trametinib,Dabrafenib, utilized to transmit signals Dinaciclib and regulate complexprocesses in cells. Protein synthesis Induces cell apoptosisL-asparaginase/Elspar ® (Merck & Co.) inhibitors ImmunotherapeuticInduces cancer patients to Alpha interferon agents exhibit immuneAngiogenesis Inhibitor/Avastin ® responsiveness (Genentech) IL-2→Interleukin 2 (Aldesleukin)/Proleukin ® (Chiron) IL-12→ Interleukin 12Antibody/small molecule Anti-CTLA-4 and PR-1 therapies immune checkpointYervoy ® (ipilimumab; Bristol-Myers Squibb) modulators Opdivo ®(nivolumab; Bristol-Myers Squibb) Keytrada ® (pembrolizumab; Merck)Hormones Hormone therapies Toremifene citrate/Fareston ® (GTX, Inc.)associated with menopause Fulvestrant/Faslodex ® (AstraZeneca) and agingseek to increase Raloxifene HCL/Evista ® (Lilly) the amount of certainAnastrazole/Arimidex ® (AstraZeneca) hormones in your body toLetrozole/Femara ® (Novartis) compensate for age- or Fadrozole (CGS16949A) disease-related hormonal Exemestane/Aromasin ® (Pharmacia &declines. Hormone therapy Upjohn) as a cancer treatment eitherLeuprolide acetate/Eligard ® (QTL USA) reduces the level of specificLupron ® (TAP Pharm) hormones or alters the Goserelin acetate/Zoladex ®(AstraZeneca) cancer's ability to use these Triptorelinpamoate/Trelstar ® (Watson Labs) hormones to grow andBuserelin/Suprefact ® (Sanofi Aventis) spread. Nafarelin/Synarel ®(Pfizer) Cetrorelix/Cetrotide ® (EMD Serono) Bicalutamide/Casodex ®(AstraZeneca) Nilutamide/Nilandron ® (Aventis Pharm.) Megestrolacetate/Megace ® (BMS) Somatostatin Analogs (Octreotide acetate/Sandostatin ® (Novartis) Glucocorticoids Anti-inflammatory drugsPrednisolone used to reduce swelling that Dexamethasone/Decadron ®(Wyeth) causes cancer pain. Aromatose inhibitors Includes imidazolesKetoconazole mTOR inhibitors the mTOR signaling Sirolimus(Rapamycin)/Rapamune ® (Wyeth) pathway was originally Temsirolimus(CCI-779)/Torisel ® (Wyeth) discovered during studies of Deforolimus(AP23573)/(Ariad Pharm.) the immunosuppressive Everolimus(RAD00I)/Certican ® (Novartis) agent rapamycin. This highly conservedpathway regulates cell proliferation and metabolism in response toenvironmental factors, linking cell growth factor receptor signaling viaphosphoinositide-3- kinase(PI-3K) to cell growth, proliferation, andangiogenesis.

In addition to the above anti-cancer agents, the anti-B7-H3 antibodiesand ADCs described herein may be administered in combination with theagents described herein. Further, the aforementioned anti-cancer agentsmay also be used in the ADCs of the invention.

In particular embodiments, the anti-B7-H3 antibodies or ADCs can beadministered alone or with another anti-cancer agent which acts inconjunction with or synergistically with the antibody to treat thedisease associated with B7-H3 activity. Such anti-cancer agents include,for example, agents well known in the art (e.g., cytotoxins,chemotherapeutic agents, small molecules and radiation). Examples ofanti-cancer agents include, but are not limited to, Panorex(Glaxo-Welcome), Rituxan (IDEC/Genentech/Hoffman la Roche). Mylotarg(Wyeth), Campath (Millennium), Zevalin (IDEC and Schering AG), Bexxar(Corixa/GSK), Erbitux (Imclone/BMS), Avastin (Genentech) and Herceptin(Genentech/Hoffman la Roche). Other anti-cancer agents include, but arenot limited to, those disclosed in U.S. Pat. No. 7,598,028 andInternational Publication No. WO2008/100624, the contents of which arehereby incorporated by reference. One or more anti-cancer agents may beadministered either simultaneously or before or after administration ofan antibody or antigen binding portion thereof of the invention.

In particular embodiments of the invention, the anti-B7-H3 antibodies orADCs described herein can be used in a combination therapy with anapoptotic agent, such as a Bcl-xL inhibitor or a Bcl-2 (B-cell lymphoma2) inhibitor (e.g., ABT-199 (venetoclax)) to treat cancer, such asleukemia, in a subject. In one embodiment, the anti-B7-H3 antibodies orADCs described herein can be used in a combination therapy with a Bcl-xLinhibitor for treating cancer. In one embodiment, the anti-B7-H3antibodies or ADCs described herein can be used in a combination therapywith venetoclax for treating cancer.

In particular embodiments of the invention, the anti-B7-H3 antibodies orADCs described herein can be used in a combination therapy with aninhibitor of NAMPT (see examples of inhibitors in US 2013/0303509;AbbVie. Inc., incorporated by reference herein) to treat a subject inneed thereof. NAMPT (also known as pre-B-cell-colony-enhancing factor(PBEF) and visfatin) is an enzyme that catalyzes the phosphoribosylationof nicotinamide and is the rate-limiting enzyme in one of two pathwaysthat salvage NAD. In one embodiment of the invention, anti-B7-H3antibodies and ADCs described herein are administered in combinationwith a NAMPT inhibitor for the treatment of cancer in a subject.

In particular embodiments of the invention, the anti-B7-H3 antibodies orADCs described herein can be used in a combination therapy with SN-38,which is the active metabolite of the topoisomerase inhibitoririnotecan.

In other embodiments of the invention, the anti-B7-H3 antibodies or ADCsdescribed herein can be used in a combination therapy with a PARP (polyADP ribose polymerase) inhibitor, e.g., veliparib, to treat cancer,including breast, ovarian and non-small cell lung cancers.

Further examples of additional therapeutic agents that can beco-administered and/or formulated with anti-B7-H3 antibodies oranti-B7-H3 ADCs described herein, include, but are not limited to, oneor more of: inhaled steroids; beta-agonists, e.g., short-acting orlong-acting beta-agonists; antagonists of leukotrienes or leukotrienereceptors; combination drugs such as ADVAIR; IgE inhibitors, e.g.,anti-IgE antibodies (e.g., XOLAIR®, omalizumab); phosphodiesteraseinhibitors (e.g., PDE4 inhibitors); xanthines; anticholinergic drugs;mast cell-stabilizing agents such as cromolyn; IL-4 inhibitors; IL-5inhibitors; eotaxin/CCR3 inhibitors; antagonists of histamine or itsreceptors including H1, H2, H3, and H4, and antagonists of prostaglandinD or its receptors (DPI and CRTH2). Such combinations can be used totreat, for example, asthma and other respiratory disorders. Otherexamples of additional therapeutic agents that can be co-administeredand/or formulated with anti-B7-H3 antibodies or anti-B7-H3 ADCsdescribed herein, include, but are not limited to, one or more of, ananti-PD1 antibody (e.g., pembrolizumab), an anti-PD-L1 antibody (e.g.,atezolizumab), an anti-CTLA-4 antibody (e.g., ipilimumab), a MEKinhibitor (e.g., trametinib), an ERK inhibitor, a BRAF inhibitor (e.g.,dabrafenib), osimertinib (AZD9291), erlotinib, gefitinib, sorafenib, aCDK9 inhibitor (e.g., dinaciclib), a MCL-1 inhibitor, temozolomide, aBcl-xL inhibitor, a Bcl-2 inhibitor (e.g., venetoclax), ibrutinib, amTOR inhibitor (e.g., everolimus), a PI3K inhibitor (e.g., buparlisib),duvelisib, idelalisib, an AKT inhibitor, a HER2 inhibitor (e.g.,lapatinib), Herceptin, a taxane (e.g., docetaxel, paclitaxel,nab-paclitaxel), an ADC comprising an auristatin, an ADC comprising aPBD (e.g., rovalpituzumab tesirine), an ADC comprising a maytansinoid(e.g., TDM1), a TRAIL agonist, a proteasome inhibitor (e.g.,bortezomib), and a nicotinamide phosphoribosyltransferase (NAMPT)inhibitor. Additional examples of therapeutic agents that can beco-administered and/or formulated with one or more anti-B7-H3 antibodiesor fragments thereof include one or more of: TNF antagonists (e.g., asoluble fragment of a TNF receptor, e.g., p55 or p75 human TNF receptoror derivatives thereof, e.g., 75 kD TNFR-IgG (75 kD TNF receptor-IgGfusion protein, ENBREL)); TNF enzyme antagonists, e.g., TNF convertingenzyme (TACE) inhibitors; muscarinic receptor antagonists; TGF-betaantagonists: interferon gamma; perfenidone: chemotherapeutic agents,e.g., methotrexate, leflunomide, or a sirolimus (rapamycin) or an analogthereof, e.g., CCI-779; COX2 and cPLA2 inhibitors; NSAIDs;immunomodulators; p38 inhibitors, TPL-2, MK-2 and NFkB inhibitors, amongothers.

Other preferred combinations are cytokine suppressive anti-inflammatorydrug(s) (CSAIDs); antibodies to or antagonists of other human cytokinesor growth factors, for example, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-15, IL-16, IL-18, IL-21, IL-31, interferons, EMAP-II,GM-CSF, FGF, EGF, PDGF, and edothelin-1, as well as the receptors ofthese cytokines and growth factors.

Antibodies of the invention, or antigen binding portions thereof, can becombined with antibodies to cell surface molecules such as CD2, CD3,CD4, CD8, CD25. CD28, CD30, CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2),CD90. CTLA, CTLA-4. PD-1, or their ligands including CD154 (gp39 orCD40L).

Preferred combinations of therapeutic agents may interfere at differentpoints in the inflammatory cascade: preferred examples include TNFantagonists like chimeric, humanized or human TNF antibodies,adalimumab, (HUMIRA: D2E7; PCT Publication No. WO 97/29131 and U.S. Pat.No. 6,090,382, incorporated by reference herein), CA2 (Remicade™), CDP571, and soluble p55 or p75 TNF receptors, derivatives, thereof,(p75TNFR1gG (Enbrel™) or p55TNFR1gG (Lenercept), and also TNF convertingenzyme (TACE) inhibitors; similarly IL-1 inhibitors(Interleukin-1-converting enzyme inhibitors, IL-IRA etc.) may beeffective for the same reason. Other preferred combinations includeInterleukin 4.

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of an antibody or antibody portion of the invention. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result. A therapeutically effective amount of the antibodyor antibody portion may be determined by a person skilled in the art andmay vary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of the antibody or antibodyportion to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the antibody, or antibody portion, are outweighedby the therapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typically,since a prophylactic dose is used in subjects prior to or at an earlierstage of disease, the prophylactically effective amount will be lessthan the therapeutically effective amount.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage.

Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of active compound calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the active compound and the particulartherapeutic or prophylactic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an ADC, an antibody or antibodyportion of the invention is 0.1-20 mg/kg, more preferably 1-10 mg/kg. Inone embodiment, the dose of the antibody or ADC described herein is 1 to6 mg/kg, including the individual doses recited therein. e.g., 1 mg/kg,2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, and 6 mg/kg. In another embodiment,the dose of the antibody or ADC described herein is 1 to 200 μg/kg,including the individual doses recited therein. e.g., 1 μg/kg, 2 μg/kg,3 μg/kg, 4 g/kg, 5 μg/kg, 10 μg/kg, 20 μg/kg, 30 μg/kg, 40 μg/kg, 50μg/kg, 60 μg/kg, 80 μg/kg, 100 μg/kg, 120 μg/kg, 140 μg/kg, 160 g/kg,180 μg/kg and 200 μg/kg. It is to be noted that dosage values may varywith the type and severity of the condition to be alleviated. It is tobe further understood that for any particular subject, specific dosageregimens should be adjusted over time according to the individual needand the professional judgment of the person administering or supervisingthe administration of the compositions, and that dosage ranges set forthherein are exemplary only and are not intended to limit the scope orpractice of the claimed composition.

In one embodiment, an anti-B7-H3 ADC, including an ADC comprisingantibody huAbl3v1, huAb3v2.5, or huAb3v2.6, is administered to a subjectin need thereof, e.g., a subject having cancer, at a dose of 0.1 to 30mg/kg. In another embodiment, the anti-B7-H3 antibody, e.g., huAbl3v1,huAb3v2.5, huAb3v2.6, or an antigen binding portion thereof, isadministered to a subject in need thereof, e.g., a subject havingcancer, as an ADC at a dose of 1 to 15 mg/kg. In another embodiment, theanti-B7-H3 antibody. e.g., huAbl3v1, huAb3v2.5, huAb3v2.6, or an antigenbinding portion thereof, is administered to a subject in need thereof.e.g., a subject having cancer, as an ADC at a dose of 1 to 10 mg/kg. Inanother embodiment, the anti-B7-H3 antibody, e.g., huAbl3v1, huAb3v2.5,huAb3v2.6, or an antigen binding portion thereof, is administered to asubject in need thereof, e.g., a subject having cancer, as an ADC at adose of 2 to 3. In another embodiment, the anti-B7-H3 antibody, e.g.,huAbl3v1, huAb3v2.5, huAb3v2.6, or an antigen binding portion thereof,is administered to a subject in need thereof, e.g., a subject havingcancer, as an ADC at a dose of 1 to 4 mg/kg.

In one embodiment, an anti-B7-H3 antibody or ADC described herein, e.g.,huAbl3v1, huAb3v2.5, huAb3v2.6, is administered to a subject in needthereof, e.g., a subject having cancer, as an ADC at a dose of 1 to 200μg/kg. In another embodiment, the anti-B7-H3 antibody. e.g., huAbl3v1,huAb3v2.5, huAb3v2.6, or an antigen binding portion thereof, isadministered to a subject in need thereof, e.g., a subject havingcancer, as an ADC at a dose of 5 to 150 μg/kg. In another embodiment,the anti-B7-H3 antibody, e.g., huAbl3v1, huAb3v2.5, huAb3v2.6, or anantigen binding portion thereof, is administered to a subject in needthereof. e.g., a subject having cancer, as an ADC at a dose of 5 to 100μg/kg. In another embodiment, the anti-B7-H3 antibody, e.g., huAbl3v1,huAb3v2.5, huAb3v2.6, or an antigen binding portion thereof, isadministered to a subject in need thereof, e.g., a subject havingcancer, as an ADC at a dose of 5 to 90 μg/kg. In another embodiment, theanti-B7-H3 antibody, e.g., huAb3v1, huAb3v2.5, huAb3v2.6, or an antigenbinding portion thereof, is administered to a subject in need thereof,e.g., a subject having cancer, as an ADC at a dose of 5 to 80 μg/kg. Inanother embodiment, the anti-B7-H3 antibody. e.g., huAbl3v1, huAb3v2.5,huAb3v2.6, or an antigen binding portion thereof, is administered to asubject in need thereof, e.g., a subject having cancer, as an ADC at adose of 5 to 70 μg/kg. In another embodiment, the anti-B7-H3 antibody,e.g., huAbl3v1, huAb3v2.5, huAb3v2.6, or an antigen binding portionthereof, is administered to a subject in need thereof, e.g., a subjecthaving cancer, as an ADC at a dose of 5 to 60 μg/kg. In anotherembodiment, the anti-B7-H3 antibody. e.g., huAbl3v1, huAb3v2.5,huAb3v2.6, or an antigen binding portion thereof, is administered to asubject in need thereof. e.g., a subject having cancer, as an ADC at adose of 10 to 80 μg/kg.

Doses described above may be useful for the administration of eitheranti-B7-H3 ADCs or antibodies disclosed herein.

In another aspect, this application provides a method for detecting thepresence of B7-H3 in a sample in vitro (e.g., a biological sample, suchas serum, plasma, tissue, and biopsy). The subject method can be used todiagnose a disorder, e.g., a cancer. The method includes: (i) contactingthe sample or a control sample with the anti-B7-H3 antibody or fragmentthereof as described herein; and (ii) detecting formation of a complexbetween the anti-B7-H3 antibody or fragment thereof, and the sample orthe control sample, wherein a statistically significant change in theformation of the complex in the sample relative to the control sample isindicative of the presence of B7-H3 in the sample.

Given their ability to bind to human B7-H3, the anti-human B7-H3antibodies, or portions thereof, of the invention, (as well as ADCsthereof) can be used to detect human B7-H3 (e.g., in a biologicalsample, such as serum or plasma), using a conventional immunoassay, suchas an enzyme linked immunosorbent assays (ELISA), an radioimmunoassay(RIA) or tissue immunohistochemistry. In one aspect, the inventionprovides a method for detecting human B7-H3 in a biological samplecomprising contacting a biological sample with an antibody, or antibodyportion, of the invention and detecting either the antibody (or antibodyportion) bound to human B7-H3 or unbound antibody (or antibody portion),to thereby detect human B7-H3 in the biological sample. The antibody isdirectly or indirectly labeled with a detectable substance to facilitatedetection of the bound or unbound antibody. Suitable detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,0-galactosidase, or acetylcholinesterase: examples of suitableprosthetic group complexes include streptavidin/biotin andavidin,biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; and examples ofsuitable radioactive material include ³H, ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In,¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, or ¹⁵³Sm.

Alternative to labeling the antibody, human B7-H3 can be assayed inbiological fluids by a competition immunoassay utilizing rhB7-H3standards labeled with a detectable substance and an unlabeledanti-human B7-H3 antibody. In this assay, the biological sample, thelabeled rhB7-H3 standards and the anti-human B7-H3 antibody are combinedand the amount of labeled rhB7-H3 standard bound to the unlabeledantibody is determined. The amount of human B7-H3 in the biologicalsample is inversely proportional to the amount of labeled rhB7-H3standard bound to the anti-B7-H3 antibody. Similarly, human B7-H3 canalso be assayed in biological fluids by a competition immunoassayutilizing rhB7-H3 standards labeled with a detectable substance and anunlabeled anti-human B7-H3 antibody.

In yet another aspect, this application provides a method for detectingthe presence of B7-H3 in vivo (e.g., in vivo imaging in a subject). Thesubject method can be used to diagnose a disorder, e.g., aB7-H3-associated disorder. The method includes: (i) administering theanti-B7-H3 antibody or fragment thereof as described herein to a subjector a control subject under conditions that allow binding of the antibodyor fragment to B7-H3; and (ii) detecting formation of a complex betweenthe antibody or fragment and B7-H3, wherein a statistically significantchange in the formation of the complex in the subject relative to thecontrol subject is indicative of the presence of B7-H3.

VI. Pharmaceutical Compositions

The invention also provides pharmaceutical compositions comprising anantibody, or antigen binding portion thereof, or ADC of the inventionand a pharmaceutically acceptable carrier. The pharmaceuticalcompositions comprising antibodies or ADCs of the invention are for usein, but not limited to, diagnosing, detecting, or monitoring a disorder,in preventing, treating, managing, or ameliorating of a disorder or oneor more symptoms thereof, and/or in research. In a specific embodiment,a composition comprises one or more antibodies of the invention. Inanother embodiment, the pharmaceutical composition comprises one or moreantibodies or ADCs of the invention and one or more prophylactic ortherapeutic agents other than antibodies or ADCs of the invention fortreating a disorder in which B7-H3 activity is detrimental. Preferably,the prophylactic or therapeutic agents known to be useful for or havingbeen or currently being used in the prevention, treatment, management,or amelioration of a disorder or one or more symptoms thereof. Inaccordance with these embodiments, the composition may further compriseof a carrier, diluent or excipient.

The antibodies and antibody-portions or ADCs of the invention can beincorporated into pharmaceutical compositions suitable foradministration to a subject. Typically, the pharmaceutical compositioncomprises an antibody or antibody portion of the invention and apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like that are physiologically compatible.Examples of pharmaceutically acceptable carriers include one or more ofwater, saline, phosphate buffered saline, dextrose, glycerol, ethanoland the like, as well as combinations thereof. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol, sorbitol, or sodium chloride in the composition.Pharmaceutically acceptable carriers may further comprise minor amountsof auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the antibody or antibody portion or ADC.

Various delivery systems are known and can be used to administer one ormore antibodies or ADCs of the invention or the combination of one ormore antibodies of the invention and a prophylactic agent or therapeuticagent useful for preventing, managing, treating, or ameliorating adisorder or one or more symptoms thereof, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the antibody or antibody fragment, receptor-mediatedendocytosis (see. e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)),construction of a nucleic acid as part of a retroviral or other vector,etc. Methods of administering a prophylactic or therapeutic agent of theinvention include, but are not limited to, parenteral administration(e.g., intradermal, intramuscular, intraperitoneal, intravenous andsubcutaneous), epidural administration, intratumoral administration, andmucosal administration (e.g., intranasal and oral routes). In addition,pulmonary administration can be employed, e.g., by use of an inhaler ornebulizer, and formulation with an aerosolizing agent. See, e.g., U.S.Pat. Nos. 6,019,968, 5,985, 320, 5,985,309, 5,934, 272, 5,874.064,5,855,913, 5.290, 540, and 4,880,078; and PCT Publication Nos. WO92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903, eachof which is incorporated herein by reference their entireties. In oneembodiment, an antibody of the invention, combination therapy, or acomposition of the invention is administered using Alkermes AIR@Apulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass.).In a specific embodiment, prophylactic or therapeutic agents of theinvention are administered intramuscularly, intravenously,intratumorally, orally, intranasally, pulmonary, or subcutaneously. Theprophylactic or therapeutic agents may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local.

In a specific embodiment, it may be desirable to administer theprophylactic or therapeutic agents of the invention locally to the areain need of treatment; this may be achieved by, for example, and not byway of limitation, local infusion, by injection, or by means of animplant, said implant being of a porous or non-porous material,including membranes and matrices, such as sialastic membranes, polymers,fibrous matrices (e.g., Tissuel®), or collagen matrices. In oneembodiment, an effective amount of one or more antibodies of theinvention antagonists is administered locally to the affected area to asubject to prevent, treat, manage, and/or ameliorate a disorder or asymptom thereof.

In another embodiment, an effective amount of one or more antibodies ofthe invention is administered locally to the affected area incombination with an effective amount of one or more therapies (e.g., oneor more prophylactic or therapeutic agents) other than an antibody ofthe invention of a subject to prevent, treat, manage, and/or amelioratea disorder or one or more symptoms thereof.

In another embodiment, the prophylactic or therapeutic agent of theinvention can be delivered in a controlled release or sustained releasesystem. In one embodiment, a pump may be used to achieve controlled orsustained release (see Langer, supra; Sefton, 1987, CRC Crit. Ref BiomedEng. 14:20; Buchwald et al., 1980. Surgery 88:507; Saudek et al., 1989,N. Engl. J. Med 321:574). In another embodiment, polymeric materials canbe used to achieve controlled or sustained release of the therapies ofthe invention (see e.g., Medical Applications of Controlled Release,Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); ControlledDrug Bioavailability, Drug Product Design and Performance. Smolen andBall (eds.), Wiley, New York (1984), Ranger and Peppas, 1983, J.,Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985,Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard etal., 1989, J. Neurosurg. 7 1:105); U.S. Pat. Nos. 5,679,377; 5,916,597;5,912,015; 5,989,463; 5,128,326; PCT Publication No. WO 99/15154; andPCT Publication No. WO 99/20253. Examples of polymers used in sustainedrelease formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid),poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides(PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol),polyacrylamide, poly(ethylene glycol), polylactides (PLA),poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In a preferredembodiment, the polymer used in a sustained release formulation isinert, free of leachable impurities, stable on storage, sterile, andbiodegradable. In yet another embodiment, a controlled or sustainedrelease system can be placed in proximity of the prophylactic ortherapeutic target, thus requiring only a fraction of the systemic dose(see, e.g., Goodson, in Medical Applications of Controlled Release,supra, vol. 2, pp. 115-138 (1984)).

Controlled release systems are discussed in the review by Langer (1990.Science 249:1527-1533). Any technique known to one of skill in the artcan be used to produce sustained release formulations comprising one ormore therapeutic agents of the invention. See, e.g., U.S. Pat. No.4,526,938, PCT publication WO 91/05548. PCT publication WO 96/20698.Ning et al., 1996, “Intratumoral Radioimmunotherapy of a Human ColonCancer Xenograft Using a Sustained-Release Gel.” Radiotherapy & Oncology39:179-189, Song et al., 1995. “Antibody Mediated Lung Targeting ofLong-Circulating Emulsions,” PDA Journal of Pharmaceutical Science &Technology 50:372-397, Cleek et al., 1997. “Biodegradable PolymericCarriers for a bFGF Antibody for Cardiovascular Application,” Pro.Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854, and Lam et al.,1997, “Microencapsulation of Recombinant Humanized Monoclonal Antibodyfor Local Delivery,” Proc. Int'l. Symp. Control Rel. Bioact. Mater.24:759-760, each of which is incorporated herein by reference in theirentireties.

In a specific embodiment, where the composition of the invention is anucleic acid encoding a prophylactic or therapeutic agent, the nucleicacid can be administered in vivo to promote expression of its encodedprophylactic or therapeutic agent, by constructing it as part of anappropriate nucleic acid expression vector and administering it so thatit becomes intracellular, e.g., by use of a retroviral vector (see U.S.Pat. No. 4,980,286), or by direct injection, or by use of microparticlebombardment (e.g., a gene gun; Biolistic, Dupont), or coating withlipids or cell-surface receptors or transfecting agents, or byadministering it in linkage to a homeobox-like peptide which is known toenter the nucleus (see, e.g., Joliot et al., 1991, Proc. Natl. Acad Sci.USA 88:1864-1868). Alternatively, a nucleic acid can be introducedintracellularly and incorporated within host cell DNA for expression byhomologous recombination.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include, but are not limited to, parenteral, e.g.,intravenous, intradermal, subcutaneous, oral, intranasal (e.g.,inhalation), transdermal (e.g., topical), transmucosal, and rectaladministration. In a specific embodiment, the composition is formulatedin accordance with routine procedures as a pharmaceutical compositionadapted for intravenous, subcutaneous, intramuscular, oral, intranasal,or topical administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection.

If the method of the invention comprises intranasal administration of acomposition, the composition can be formulated in an aerosol form,spray, mist or in the form of drops. In particular, prophylactic ortherapeutic agents for use according to the invention can beconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebulizer, with the use of a suitable propellant(e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridges(composed of, e.g., gelatin) for use in an inhaler or insufflator may beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

If the method of the invention comprises oral administration,compositions can be formulated orally in the form of tablets, capsules,cachets, gel caps, solutions, suspensions, and the like. Tablets orcapsules can be prepared by conventional means with pharmaceuticallyacceptable excipients such as binding agents (e.g., pregelatinised maizestarch, poly vinylpyrrolidone, or hydroxypropyl methylcellulose);fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogenphosphate); lubricants (e.g., magnesium stearate, talc, or silica);disintegrants (e.g., potato starch or sodium starch glycolate); orwetting agents (e.g., sodium lauryl sulphate). The tablets may be coatedby methods well-known in the art. Liquid preparations for oraladministration may take the form of, but not limited to, solutions,syrups or suspensions, or they may be presented as a dry product forconstitution with water or other suitable vehicle before use. Suchliquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives, or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring, and sweetening agents as appropriate. Preparations for oraladministration may be suitably formulated for slow release, controlledrelease, or sustained release of a prophylactic or therapeutic agent(s).

The method of the invention may comprise pulmonary administration, e.g.,by use of an inhaler or nebulizer, of a composition formulated with anaerosolizing agent. See. e.g., U.S. Pat. Nos. 6,019,968, 5,985,320,5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078;and PCT Publication Nos. WO 92/19244, WO 97/32572, WO 9744013, WO98/31346, and WO 99/66903, each of which is incorporated herein byreference their entireties. In a specific embodiment, an antibody of theinvention, combination therapy, and/or composition of the invention isadministered using Alkermes AIR pulmonary drug delivery technology(Alkermes, Inc., Cambridge, Mass.).

The method of the invention may comprise administration of a compositionformulated for parenteral administration by injection (e.g., by bolusinjection or continuous infusion). Formulations for injection may bepresented in unit dosage form (e.g., in ampoules or in multi-dosecontainers) with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle (e.g., sterile pyrogen-free water) before use.

The methods of the invention may additionally comprise of administrationof compositions formulated as depot preparations. Such long actingformulations may be administered by implantation (e.g., subcutaneouslyor intramuscularly) or by intramuscular injection. Thus, for example,the compositions may be formulated with suitable polymeric orhydrophobic materials (e.g., as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives (e.g., as asparingly soluble salt).

The methods of the invention encompass administration of compositionsformulated as neutral or salt forms. Pharmaceutically acceptable saltsinclude those formed with anions such as those derived fromhydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., andthose formed with cations such as those derived from sodium, potassium,ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine,2-ethylamino ethanol, histidine, procaine, etc.

Generally, the ingredients of compositions are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the mode of administration is infusion, compositioncan be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the mode of administrationis by injection, an ampoule of sterile water for injection or saline canbe provided so that the ingredients may be mixed prior toadministration.

In particular, the invention also provides that one or more of theprophylactic or therapeutic agents, or pharmaceutical compositions ofthe invention is packaged in a hermetically sealed container such as anampoule or sachette indicating the quantity of the agent. In oneembodiment, one or more of the prophylactic or therapeutic agents, orpharmaceutical compositions of the invention is supplied as a drysterilized lyophilized powder or water free concentrate in ahermetically sealed container and can be reconstituted (e.g., with wateror saline) to the appropriate concentration for administration to asubject. Preferably, one or more of the prophylactic or therapeuticagents or pharmaceutical compositions of the invention is supplied as adry sterile lyophilized powder in a hermetically sealed container at aunit dosage of at least 5 mg, at least 10 mg, at least 15 mg, at least25 mg, at least 35 mg, at least 45 mg, at least 50 mg, at least 75 mg,or at least 100 mg. The lyophilized prophylactic or therapeutic agentsor pharmaceutical compositions of the invention should be stored atbetween 2° C., and 8° C. in its original container and the prophylacticor therapeutic agents, or pharmaceutical compositions of the inventionshould be administered within 1 week, within 5 days, within 72 hours,within 48 hours, within 24 hours, within 12 hours, within 6 hours,within 5 hours, within 3 hours, or within 1 hour after beingreconstituted. In an alternative embodiment, one or more of theprophylactic or therapeutic agents or pharmaceutical compositions of theinvention is supplied in liquid form in a hermetically sealed containerindicating the quantity and concentration of the agent. Preferably, theliquid form of the administered composition is supplied in ahermetically sealed container at least 0.25 mg/ml, at least 0.5 mg/ml,at least 1 mg/ml, at least 2.5 mg/ml, at least 5 mg/ml, at least 8mg/ml, at least 10 mg/ml, at least 15 mg/kg, at least 25 mg/ml, at least50 mg/ml, at least 75 mg/ml or at least 100 mg/ml. The liquid formshould be stored at between 2° C., and 8° C. in its original container.

The antibodies and antibody-portions of the invention can beincorporated into a pharmaceutical composition suitable for parenteraladministration. Preferably, the antibody or antibody-portions will beprepared as an injectable solution containing 0.1-250 mg/ml antibody.The injectable solution can be composed of either a liquid orlyophilized dosage form in a flint or amber vial, ampule or pre-filledsyringe. The buffer can be L-histidine (1-50 mM), optimally 5-10 mM, atpH 5.0 to 7.0 (optimally pH 6.0). Other suitable buffers include but arenot limited to, sodium succinate, sodium citrate, sodium phosphate orpotassium phosphate. Sodium chloride can be used to modify the toxicityof the solution at a concentration of 0-300 mM (optimally 150 mM for aliquid dosage form). Cryoprotectants can be included for a lyophilizeddosage form, principally 0-10%/sucrose (optimally 0.5-1.0%). Othersuitable cryoprotectants include trehalose and lactose. Bulking agentscan be included for a lyophilized dosage form, principally 1-10%mannitol (optimally 24%).

Stabilizers can be used in both liquid and lyophilized dosage forms,principally 1-50 mM L-methionine (optimally 5-10 mM). Other suitablebulking agents include glycine, arginine, can be included as 0-0.05%polysorbate-80 (optimally 0.005-0.01%). Additional surfactants includebut are not limited to polysorbate 20 and BRIJ surfactants. Thepharmaceutical composition comprising the antibodies andantibody-portions of the invention prepared as an injectable solutionfor parenteral administration, can further comprise an agent useful asan adjuvant, such as those used to increase the absorption, ordispersion of a therapeutic protein (e.g., antibody). A particularlyuseful adjuvant is hyaluronidase, such as Hylenex® (recombinant humanhyaluronidase). Addition of hyaluronidase in the injectable solutionimproves human bioavailability following parenteral administration,particularly subcutaneous administration. It also allows for greaterinjection site volumes (i.e. greater than 1 ml) with less pain anddiscomfort, and minimum incidence of injection site reactions. (seeWO2004078140, US2006104968 incorporated herein by reference).

The compositions of this invention may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, liposomes and suppositories.The preferred form depends on the intended mode of administration andtherapeutic application. Typical preferred compositions are in the formof injectable or infusible solutions, such as compositions similar tothose used for passive immunization of humans with other antibodies. Thepreferred mode of administration is parenteral (e.g., intravenous,subcutaneous, intraperitoneal, intramuscular). In a preferredembodiment, the antibody is administered by intravenous infusion orinjection. In another preferred embodiment, the antibody is administeredby intramuscular or subcutaneous injection.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the active compound (i.e.,antibody or antibody portion) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile,lyophilized powders for the preparation of sterile injectable solutions,the preferred methods of preparation are vacuum drying and spray-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The proper fluidity of a solution can be maintained, for example, by theuse of a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding, in the composition, an agent that delays absorption, forexample, monostearate salts and gelatin.

The antibodies and antibody-portions or ADCs of the invention can beadministered by a variety of methods known in the art, although for manytherapeutic applications, the preferred route/mode of administration issubcutaneous injection, intravenous injection or infusion. As will beappreciated by the skilled artisan, the route and/or mode ofadministration will vary depending upon the desired results. In certainembodiments, the active compound may be prepared with a carrier thatwill protect the compound against rapid release, such as a controlledrelease formulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See. e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

In certain embodiments, an antibody or antibody portion or ADC of theinvention may be orally administered, for example, with an inert diluentor an assimilable edible carrier. The compound (and other ingredients,if desired) may also be enclosed in a hard or soft shell gelatincapsule, compressed into tablets, or incorporated directly into thesubject's diet. For oral therapeutic administration, the compounds maybe incorporated with excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. To administer a compound of the inventionby other than parenteral administration, it may be necessary to coat thecompound with, or co-administer the compound with, a material to preventits inactivation.

In other embodiments, an antibody or antibody portion or ADC of theinvention may be conjugated to a polymer-based species such that saidpolymer-based species may confer a sufficient size upon said antibody orantibody portion of the invention such that said antibody or antibodyportion of the invention benefits from the enhanced permeability andretention effect (EPR effect) (See also PCT Publication No.WO2006/042146A2 and U.S. Publication Nos. 2004/0028687A, 2009/0285757A,and 2011/0217363A1, and U.S. Pat. No. 7,695,719 (each of which isincorporated by reference herein in its entirety and for all purposes).

Supplementary active compounds can also be incorporated into thecompositions. In certain embodiments, an antibody or antibody portion orADC of the invention is formulated with and/or co-administered with oneor more additional therapeutic agents that are useful for treatingdisorders in which B7-H3 activity is detrimental. For example, ananti-hB7-H3 antibody or antibody portion or ADC of the invention may beformulated and/or co-administered with one or more additional antibodiesthat bind other targets (e.g., antibodies that bind cytokines or thatbind cell surface molecules). Furthermore, one or more antibodies of theinvention may be used in combination with two or more of the foregoingtherapeutic agents. Such combination therapies may advantageouslyutilize lower dosages of the administered therapeutic agents, thusavoiding possible toxicities or complications associated with thevarious monotherapies.

In certain embodiments, an antibody or ADC to B7-H3 or fragment thereofis linked to a half-life extending vehicle known in the art. Suchvehicles include, but are not limited to, the Fc domain, polyethyleneglycol, and dextran. Such vehicles are described. e.g., in U.S.application Ser. No. 09/428,082 and published PCT Application No. WO99/25044, which are hereby incorporated by reference for any purpose.

It will be readily apparent to those skilled in the art that othersuitable modifications and adaptations of the methods of the inventiondescribed herein are obvious and may be made using suitable equivalentswithout departing from the scope of the invention or the embodimentsdisclosed herein. Having now described the invention in detail, the samewill be more clearly understood by reference to the following examples,which are included for purposes of illustration only and are notintended to be limiting

EXAMPLES Example 1: Synthesis of Exemplary Bcl-xL Inhibitors

This example provides synthetic methods for exemplary Bcl-xL inhibitorycompounds W2.01-W2.91. Bcl-xL inhibitors (W2.01-W2.91) and synthons(Examples 2.1-2.176) were named using ACD/Name 2012 release (Build56084, 5 Apr. 2012. Advanced Chemistry Development Inc. Toronto,Ontario), ACD/Name 2014 release (Build 66687, 25 Oct. 2013, AdvancedChemistry Development Inc., Toronto, Ontario), ChemDraw® Ver. 9.0.7(CambridgeSoft, Cambridge, Mass.), ChemDraw® Ultra Ver. 12.0(CambridgeSoft. Cambridge, Mass.), or ChemDraw® Professional Ver.15.0.0.106. Bcl-xL inhibitor and synthon intermediates were named withACD/Name 2012 release (Build 56084, 5 Apr. 2012. Advanced ChemistryDevelopment Inc., Toronto, Ontario), ACD/Name 2014 release (Build 66687,25 Oct. 2013, Advanced Chemistry Development Inc., Toronto, Ontario),ChemDraw® Ver. 9.0.7 (CambridgeSoft, Cambridge, Mass.), ChemDraw-UltraVer. 12.0 (CambridgeSoft. Cambridge, Mass.), or ChemDraw® ProfessionalVer. 15.0.0.106.

1.1 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-[1-({3-[2-({2-[2-(carboxymethoxy)ethoxy]ethyl}amino)ethoxy]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylicAcid (Compound W2.01) 1.1.1 3-bromo-5,7-dimethyladamantanecarboxylicAcid

Into a 50 mL round-bottomed flask at 0° C., was added bromine (16 mL).Iron powder (7 g) was added, and the reaction was stirred at 0° C. for30 minutes. 35-Dimethyladamantane-1-carboxylic acid (12 g) was added.The mixture was warmed up to room temperature and stirred for 3 days. Amixture of ice and concentrated HCl was poured into the reactionmixture. The resulting suspension was treated twice with Na₂SO₃ (50 g in200 mL water) and extracted three times with dichloromethane. Thecombined organics were washed with 1N aqueous HCl, dried over sodiumsulfate, filtered, and concentrated to give the title compound.

1.1.2 3-bromo-5,7-dimethyladamantanemethanol

To a solution of Example 1.1.1 (15.4 g) in tetrahydrofuran (200 mL) wasadded BH₃ (1M in tetrahydrofuran, 150 mL), and the mixture was stirredat room temperature overnight. The reaction mixture was then carefullyquenched by adding methanol dropwise. The mixture was then concentratedunder vacuum, and the residue was balanced between ethyl acetate (500mL) and 2N aqueous HCl (100 mL). The aqueous layer was further extractedtwice with ethyl acetate, and the combined organic extracts were washedwith water and brine, dried over sodium sulfate, and filtered.Evaporation of the solvent gave the title compound.

1.1.31-((3-bromo-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl)-1H-pyrazole

To a solution of Example 1.1.2 (8.0 g) in toluene (60 mL) was added1H-pyrazole (1.55 g) and cyanomethylenetributylphosphorane (2.0 g), andthe mixture was stirred at 90° C. overnight. The reaction mixture wasconcentrated, and the residue was purified by silica gel columnchromatography (10:1 heptane:ethyl acetate) to give the title compound.MS (ESI) m/e 324.2 (M+H)⁺.

1.1.42-{[3,5-dimethyl-7-(1H-pyrazol-1-ylmethyl)tricyclo[3.3.1.1^(3,7)]dec-1-yl]oxy}ethanol

To a solution of Example 1.1.3 (4.0 g) in ethane-1,2-diol (12 mL) wasadded triethylamine (3 mL). The mixture was stirred at 150° C. undermicrowave conditions (Biotage Initiator) for 45 minutes. The mixture waspoured into water (100 mL) and extracted three times with ethyl acetate.The combined organic extracts were washed with water and brine, driedover sodium sulfate, and filtered. Evaporation of the solvent gave aresidue that was purified by silica gel chromatography, eluting with 20%ethyl acetate in heptane, followed by 5% methanol in dichloromethane, togive the title compound. MS (ESI) m/e 305.2 (M+H)⁺.

1.1.52-({3,5-dimethyl-7-[(5-methyl-1H-pyrazol-1-yl)methyl]tricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethanol

To a cooled (−78° C.) solution of Example 1.1.4 (6.05 g) intetrahydrofuran (100 mL) was added n-BuLi (40 mL. 2.5M in hexane), andthe mixture was stirred at −78° C. for 1.5 hours. Iodomethane (10 mL)was added through a syringe, and the mixture was stirred at −78° C. for3 hours. The reaction mixture was then quenched with aqueous NH₄Cl andextracted twice with ethyl acetate, and the combined organic extractswere washed with water and brine. After drying over sodium sulfate, thesolution was filtered and concentrated, and the residue was purified bysilica gel column chromatography, eluting with 5% methanol indichloromethane, to give the title compound. MS (ESI) m/e 319.5 (M+H)⁺.

1.1.61-({3,5-dimethyl-7-[2-(hydroxy)ethoxy]tricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-4-iodo-5-methyl-1H-pyrazole

To a solution of Example 1.1.5 (3.5 g) in N,N-dimethylformamide (30 mL)was added N-iodosuccinimide (3.2 g), and the mixture was stirred at roomtemperature for 1.5 hours. The reaction mixture was diluted with ethylacetate (600 mL) and washed with aqueous NaHSO₃, water and brine. Theorganic layer was dried over sodium sulfate, filtered and concentratedunder reduced pressure. The residue was purified by silica gelchromatography, eluting with 20/o ethyl acetate in dichloromethane, togive the title compound. MS (ESI) m/e 445.3 (M+H)⁺.

1.1.71-((3-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-4-iodo-5-methyl-1H-pyrazole

Tert-butyldimethylsilyl trifluoromethanesulfonate (5.34 mL) was added toa solution of Example 1.1.6 (8.6 g) and 2,6-lutidine (3.16 mL) indichloromethane (125 mL) at −40° C., and the reaction was allowed towarm to room temperature overnight. The mixture was concentrated, andthe residue was purified by silica gel chromatography, eluting with5-20% ethyl acetate in heptanes, to give the title compound. MS (ESI)m/e 523.4 (M+H)⁺.

1.1.81-((3-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole

n-Butyllithium (8.42 mL. 2.5M in hexanes) was added to Example 1.1.7(9.8 g) in 120 mL tetrahydrofuran at −78° C., and the reaction wasstirred for 1 minute. Trimethyl borate (3.92 mL) was added, and thereaction stirred for 5 minutes. Pinacol (6.22 g) was added, and thereaction was allowed to warm to room temperature and was stirred 2hours. The reaction was quenched with pH 7 buffer, and the mixture waspoured into ether. The layers were separated, and the organic layer wasconcentrated under reduced pressure. The residue was purified by silicagel chromatography, eluting with 1-25% ethyl acetate in heptanes, togive the title compound.

1.1.9 6-fluoro-3-bromopicolinic Acid

A slurry of 6-amino-3-bromopicolinic acid (25 g) in 400 mL 1:1dichloromethane/chloroform was added to nitrosonium tetrafluoroborate(18.2 g) in dichloromethane (100 mL) at 5° C. over 1 hour. The resultingmixture was stirred for another 30 minutes, then warmed to 35° C., andstirred overnight. The reaction was cooled to room temperature, and thenadjusted to pH 4 with aqueous NaH₂PO₄ solution. The resulting solutionwas extracted three times with dichloromethane, and the combinedextracts were washed with brine, dried over sodium sulfate, filtered andconcentrated to provide the title compound.

1.1.10 Tert-butyl 3-bromo-6-fluoropicolinate

Para-toluenesulfonyl chloride (27.6 g) was added to a solution ofExample 1.1.9 (14.5 g) and pyridine (26.7 mL) in dichloromethane (100mL) and tert-butanol (80 mL) at 0° C. The reaction was stirred for 15minutes, and then warmed to room temperature, and stirred overnight. Thesolution was concentrated and partitioned between ethyl acetate andaqueous Na₂CO₃ solution. The layers were separated, and the aqueouslayer extracted with ethyl acetate. The organic layers were combined,rinsed with aqueous Na₂CO₃ solution and brine, dried over sodiumsulfate, filtered, and concentrated to provide the title compound.

1.1.11 Methyl2-(5-bromo-6-(tert-butoxycarbonyl)pyridin-2-yl)-1,2,3,4-tetrahydroisoquinoline-8-carboxylate

To a solution of methyl 1,2,3,4-tetrahydroisoquinoline-8-carboxylatehydrochloride (12.37 g) and Example 1.1.10 (15 g) in dimethyl sulfoxide(100 mL) was added N,N-diisopropylethylamine (12 mL), and the mixturewas stirred at 50° C. for 24 hours. The mixture was then diluted withethyl acetate (500 mL) and washed with water and brine. The organiclayer was dried over sodium sulfate, filtered and concentrated underreduced pressure. The residue was purified by silica gel chromatography,eluting with 20% ethyl acetate in hexane, to give the title compound. MS(ESI) m/e 448.4 (M+H)⁺.

1.1.12 Methyl2-(6-(tert-butoxycarbonyl)-5-(1-((3-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)pyridin-2-yl)-1,2,3,4-tetrahydroisoquinoline-8-carboxylate

A mixture of Example 1.1.11 (3.08 g). Example 1.1.8 (5 g),tris(dibenzylideneacetone)dipalladium(0) (126 mg),1,3,5,7-tetramethyl-8-tetradecyl-2,4,6-trioxa-8-phosphaadamantane (170mg), and K₃P4 (3.65 g) in 1,4-dioxane (25 mL) and water (25 mL) washeated to 90° C. for 2 hours. The mixture was cooled and poured into 1:1diethyl ether:ethyl acetate. The layers were separated, and the organicwas washed with saturated aqueous NaH₂PO₄ solution, water (2×), andbrine. The organic layer was dried over sodium sulfate, filtered, andconcentrated. The residue was purified by silica gel chromatography,eluting with 1-25% ethyl acetate in heptanes, to give the titlecompound. MS (ESI) m/e 799.6 (M+H)⁺.

1.1.132-(6-(tert-butoxycarbonyl)-5-(1-((3-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)pyridin-2-yl)-1,2,3,4-tetrahydroisoquinoline-8-carboxylicAcid

Example 1.1.12 (5 g) and lithium hydroxide monohydrate (0.276 g) werestirred together in a solvent mixture of tetrahydrofuran (50 mL),methanol (5 mL) and water (15 mL) at 70° C. for 2 days. The reaction wascooled, acidified with 1M aqueous HCl solution, and extracted twice withethyl acetate. The combined organic layers were washed with brine, driedover sodium sulfate, filtered, and concentrated. The residue wasdissolved in dichloromethane (100 mL), cooled at −40° C. and2,6-lutidine (1.8 mL) and tert-butyldimethylsilyltrifluoromethanesulfonate (3.28 g) were added. The reaction was allowedto warm to room temperature and was stirred for 2 hours. The mixture wasdiluted with ether, and the layers were separated. The organic layer wasconcentrated. The residue was dissolved in tetrahydrofuran and treatedwith saturated aqueous K₂CO₃ solution for 1 hour. This mixture wasacidified with concentrated HCl and extracted twice with ethyl acetate.The combined organic layers were dried over sodium sulfate, filtered,and concentrated under reduced pressure. The residue was purified bysilica gel chromatography, eluting with 10-100% ethyl acetate inheptanes then 5% methanol in ethyl acetate, to give the title compound.MS (ESI) m/e 785.6 (M+H)⁺.

1.1.14 Tert-butyl6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-(2-hydroxyethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

Example 1.1.13 (970 mg), N,N-diisopropylethylamine (208 mg), and2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisouroniumhexafluorophosphate (HATU) (970 mg) were stirred in 7 mLN,N-dimethylformamide at 0° C. for 10 minutes. Benzo[d]thiazol-2-amine(278 mg) was added, and the mixture was stirred for 24 hours at 50° C.The mixture was cooled and diluted with ethyl acetate. The organic layerwas washed with water and brine, dried over sodium sulfate, filtered,and concentrated. The residue was dissolved in tetrahydrofuran (50 mL),and tetrabutyl ammonium fluoride (10 mL, 1M in tetrahydrofuran) wasadded. The reaction was stirred for 1 hour, poured into ethyl acetateand washed with pH 7 buffer and brine. The organic layer was dried oversodium sulfate, filtered, and concentrated under reduced pressure. Theresidue was purified by silica gel chromatography, eluting with 10-100%ethyl acetate in heptanes, to give the title compound. MS (ESI) m/e803.7 (M+H)⁺.

1.1.15 Tert-butyl6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3,5-dimethyl-7-(2-oxoethoxy)adamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

To an ambient solution of Example 1.1.14 (100 mg) in dichloromethane(1.3 mL) was added Dess-Martin periodinane (58.1 mg) in a singleportion. The reaction was stirred for 0.5 hours, and additionalDess-Martin periodinane (8 mg) was added. The reaction was stirred for 1hour and quenched by the addition of −10/aqueous NaOH solution anddichloromethane. The layers were separated, and the organic layer waswashed with ˜10% aqueous NaOH solution. The organic layer was dried withanhydrous sodium sulfate, filtered and concentrated under reducedpressure to a solid, which was used in the subsequent reaction withoutfurther purification. MS (ESI) m/e 801.3 (M+H)⁺.

1.1.162-(2-(2-((2-((3-((4-(6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-2-(tert-butoxycarbonyl)pyridin-3-yl)-5-methyl-1H-pyrazol-1-yl)methyl)-5,7-dimethyladamantan-1-yl)oxy)ethyl)amino)ethoxy)ethoxy)aceticAcid

To an ambient solution of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (22 mg)and Example 1.1.15 (100 mg) in methanol (1.3 mL) was added MP-CNBH₃ (65mg, 2.49 mmol/g loading). The reaction was gently shaken overnight andfiltered through a 0.4 micron filter. The crude material was purified byreverse phase HPLC using a Gilson system, eluting with 20-80%acetonitrile in water containing 0.1% v/v trifluoroacetic acid. Thedesired fractions were combined and freeze-dried to provide the titlecompound. MS (ESI) m/e 948.3 (M+H)⁺.

1.1.176-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-(2-((2-(2-(carboxymethoxy)ethoxy)ethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinicAcid

To an ambient solution of Example 1.1.16 (15 mg) in dichloromethane (1mL) was added trifluoroacetic acid (1 mL). The reaction was stirred for16 hours and then concentrated under reduced pressure. The residue waspurified by reverse phase HPLC using a Gilson system, eluting with20-80% acetonitrile in water containing 0.1% v/v trifluoroacetic acid.The desired fractions were combined and freeze-dried to provide thetitle compound. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm 12.70 (bs,2H), 8.29 (s, 1H), 8.03 (d, 1H), 7.79 (d, 1H), 7.62 (d, 1H), 7.53-7.42(m, 3H), 7.40-7.32 (m, 2H), 7.29 (s, 1H), 6.96 (d, 1H), 4.96 (bs, 2H),4.03 (s, 2H), 3.90 (t, 2H), 3.84 (s, 2H), 3.68 (t, 2H), 3.63-3.54 (m,6H), 3.17-3.04 (m, 4H), 3.00 (t, 2H), 2.10 (s, 3H), 1.45-1.40 (m, 2H),1.36-1.20 (m, 4H), 1.21-0.96 (m, 7H), 0.91-0.81 (m, 6H). MS (ESI) m/e892.3 (M+H)⁺.

1.2 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (Compound W2.02) 1.2.1 Methyl2-(6-(tert-butoxycarbonyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)-1,2,3,4-tetrahydroisoquinoline-8-carboxylate

To a solution of Example 1.1.11 (2.25 g) and[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladiun(II)(205 mg) inacetonitrile (30 mL) was added triethylamine (3 mL) and pinacolborane (2mL), and the mixture was stirred at reflux for 3 hours. The mixture wasdiluted with ethyl acetate (200 mL) and washed with water and brine. Theorganic layer was dried over sodium sulfate, filtered and concentratedunder reduced pressure. Purification of the residue by silica gelchromatography, eluting with 20% ethyl acetate in hexane, provided thetitle compound.

1.2.2 Methyl2-(6-(tert-butoxycarbonyl)-5-(1-((3-(2-hydroxyethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)pyridin-2-yl)-1,2,3,4-tetrahydroisoquinoline-8-carboxylate

To a solution of Example 1.2.1 (2.25 g) in tetrahydrofuran (30 mL) andwater (10 mL) was added Example 1.1.6 (2.0 g),1.3,5,7-tetramethyl-6-phenyl-2,4,8-trioxa-6-phosphaadamantane (329 mg),tris(dibenzylideneacetone)dipalladium(0) (206 mg) and potassiumphosphate tribasic (4.78 g). The mixture was refluxed overnight, cooledand diluted with ethyl acetate (500 mL). The resulting mixture waswashed with water and brine, and the organic layer was dried over sodiumsulfate, filtered and concentrated. The residue was purified by flashchromatography, eluting with 20% ethyl acetate in heptanes followed by5% methanol in dichloromethane, to provide the title compound.

1.2.3 Methyl2-(6-(tert-butoxycarbonyl)-5-(1-((3,5-dimethyl-7-(2-((methylsulfonyl)oxy)ethoxy)adamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)pyridin-2-yl)-1,2,3,4-tetrahydroisoquinoline-8-carboxylate

To a cold solution of Example 1.2.2 (3.32 g) in dichloromethane (100 mL)in an ice-bath was sequentially added triethylamine (3 mL) andmethanesulfonyl chloride (1.1 g). The reaction mixture was stirred atroom temperature for 1.5 hours and diluted with ethyl acetate, andwashed with water and brine. The organic layer was dried over sodiumsulfate, filtered, and concentrated to provide the title compound.

1.2.4 methyl2-(5-(1-((3-(2-azidoethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(tert-butoxycarbonyl)pyridin-2-yl)-1,2,3,4-tetrahydroisoquinoline-8-carboxylate

To a solution of Example 1.2.3 (16.5 g) in N,N-dimethylformamide (120mL) was added sodium azide (4.22 g). The mixture was heated at 80° C.for 3 hours, cooled, diluted with ethyl acetate and washed with waterand brine. The organic layer was dried over sodium sulfate, filtered,and concentrated. The residue was purified by flash chromatography,eluting with 20% ethyl acetate in heptanes, to provide the titlecompound.

1.2.52-(5-(1-((3-(2-azidoethoxy)-5,7-dimethyladamantan-1-y)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(tert-butoxycarbonyl)pyridin-2-yl)-1,2,3,4-tetrahydroisoquinoline-8-carboxylicAcid

To a solution of Example 1.2.4 (10 g) in a mixture of tetrahydrofuran(60 mL), methanol (30 mL) and water (30 mL) was added lithium hydroxidemonohydrate (1.2 g). The mixture was stirred at room temperatureovernight and neutralized with 2% aqueous HCl. The resulting mixture wasconcentrated, and the residue was dissolved in ethyl acetate (800 mL),and washed with brine. The organic layer was dried over sodium sulfate,filtered, and concentrated to provide the title compound.

1.2.6 Tert-butyl3-(1-((3-(2-azidoethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinate

A mixture of Example 1.2.5 (10 g), benzo[d]thiazol-2-amine (3.24 g),fluoro-N,N,N′,N′-tetramethylformamidinium hexafluorophosphate (5.69 g)and N,N-diisopropylethylamine (5.57 g) in N,N-dimethylfornamide (20 mL)was heated at 60° C. for 3 hours, cooled and diluted with ethyl acetate.The resulting mixture was washed with water and brine. The organic layerwas dried over sodium sulfate, filtered, and concentrated. The residuewas purified by flash chromatography, eluting with 20% ethyl acetate indichloromethane to give the title compound.

1.2.7 Tert-butyl3-(1-((3-(2-aminoethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinate

To a solution of Example 1.2.6 (2.0 g) in tetrahydrofuran (30 mL) wasadded Pd/C (10%, 200 mg). The mixture was stirred under a hydrogenatmosphere overnight. The insoluble material was filtered off and thefiltrate was concentrated to provide the title compound.

1.2.8 Tert-butyl6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-[1-({3,5-dimethyl-7-[(2,2,7,7-tetramethyl-10,10-dioxido-3,3-diphenyl-4,9-dioxa-10λ⁶-thia-13-aza-3-silapentadecan-15-yl)oxy]tricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylate

To a solution of Example 1.2.7 (500 mg) in N,N-dimethylformamide (8 mL)was added 4-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylbutylethenesulfonate (334 mg). The reaction was stirred at room temperatureovernight and methylamine (0.3 mL) was added to quench the reaction. Theresulting mixture was stirred for 20 minutes and purified byreverse-phase chromatography using an Analogix system (C18 column),eluting with 50-100% acetonitrile in water containing 0.1% v/vtrifluoroacetic acid, to provide the title compound.

1.2.96-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid

Example 1.2.8 (200 mg) in dichloromethane (5 mL) was treated withtrifluoroacetic acid (2.5 mL) overnight. The reaction mixture wasconcentrated and purified by reverse phase chromatography (C18 column),eluting with 20-60% acetonitrile in water containing 0.1% v/vtrifluoroacetic acid, to provide the title compound. ¹H NMR (500 MHz,dimethyl sulfoxide-d₆) δ ppm 12.86 (s, 1H), 8.32 (s, 2H), 8.02 (d, 1H),7.78 (d, 1H), 7.60 (d, 1H), 7.51 (d, 1H), 7.40-7.49 (m, 2H), 7.31-7.39(m, 2H), 7.27 (s, 1H), 6.95 (d, 1H), 4.94 (s, 2H), 3.87 (t, 2H), 3.81(s, 2H), 3.15-3.25 (m, 2H), 3.03-3.13 (m, 2H), 3.00 (t, 2H), 2.79 (t,2H), 2.09 (s, 3H), 1.39 (s, 2H), 1.22-1.34 (m, 4H), 0.94-1.18 (m, 6H),0.85 (s, 6H). MS (ESI) m/e 854.1 (M+H)⁺.

1.3 Synthesis of2-{[(2-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]amino}ethyl)sulfonyl]amino}-2-deoxy-D-glucopyranose(Compound W2.03) 1.3.13-(1-((3-(2-aminoethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid

Example 1.2.7 (200 mg) in dichloromethane (2.5 mL) was treated withtrifluoroacetic acid (2.5 mL) overnight. The reaction mixture wasconcentrated, and the residue was purified by reverse phasechromatography (C18 column), eluting with 20-60% acetonitrile in watercontaining 0.1% v/v trifluoroacetic acid, to provide the title compound.MS (ESI) me 746.2 (M+H)⁺.

1.3.2(3R,4R,5S,6R)-6-(acetoxymethyl)-3-(vinylsulfonamido)tetrahydro-2H-pyran-2,4,5-triylTriacetate

To a suspension of(3R,4R,5S,6R)-6-(acetoxymethyl)-3-aminotetrahydro-2H-pyran-2,4,5-triyltriacetate (7.7 g) in dichloromethane (100 mL) at 0° C. was added2-chloroethanesulfonyl chloride (4.34 g). The mixture was stirred at 0°C. for 15 minutes, and triethylamine (12.1 mL) was added. The mixturewas stirred at 0° C. for 1 hour, warmed to room temperature and stirredfor 2 days. The mixture was diluted with dichloromethane and washed withwater and brine. The organic layer was dried over sodium sulfate,filtered, and concentrated to provide the title compound.

1.3.3N-((3R,4R,5S,6R)-2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)ethenesulfonamide

To a solution of Example 1.3.2 (6.74 g) in methanol (150 mL) was addedtriethylamine (10 mL). The mixture was stirred for 4 days andconcentrated. The residue was dissolved in methanol and treated withDowex HCR-5 until the solution was neutral. The mixture was filtered,and the filtrate was concentrated. The residue was purified bychromatography using a column of Sephadex LH-20 (100 g), eluting withmethanol to provide the title compound.

1.3.42-{[(2-{[2-(3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]amino}ethyl)sulfonyl]amino)-2-deoxy-D-glucopyranose

A mixture of Example 1.3.1 (23.5 mg), Example 1.3.3 (42.4 mg), andN,N-diisopropylethylamine (55 τL) in N,N-dimethylformamide (1 mL) andwater (0.3 mL) was stirred for 5 days. The mixture was purified byreverse phase chromatography (C18 column), eluting with 20-60%acetonitrile in water containing 0.1% v/v trifluoroacetic acid, toprovide the title compound. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δppm 12.85 (s, 1H), 8.42 (s, 1H), 8.42 (s, 1H), 8.03 (d, 1H), 7.79 (d,1H), 7.55-7.66 (m, 1H), 7.46-7.54 (m, 2H), 7.42-7.47 (m, 1H), 7.33-7.40(m, 2H), 7.29 (s. 1H), 6.96 (d, 1H), 4.96 (s, 2H), 3.89 (t, 2H), 3.83(s, 2H), 2.97-3.14 (m, 6H), 2.10 (s, 3H), 1.44 (s. 2H), 1.22-1.39 (m,4H), 0.97-1.20 (m, 6H), 0.87 (s, 6H). MS (ESI) m/e 1015.3 (M+H)⁺.

1.4 This Paragraph was Intentionally Left Blank 1.5 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(4-{[(3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl]methyl}benzyl)amino]ethoxy}tricyclo[3.3.1.13,7]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicacid (Compound W2.05) 1.5.1[4-((3S,4R,5R,6R)-3,4,5-Tris-methoxymethoxy-6-methoxymethoxymethyl-tetrahydro-pyran-2-ylmethyl)-phenyl]-methanol

The title compound was prepared according to J. R. Walker et al.,Bioorg. Med. Chem. 2006,14,3038-3048. MS (ESI) m/e 478 (M+NH₄)⁺.

1.5.24-((3S,4R,5R,6R)-3,45-Tris-methoxymethoxy-6-methoxymethoxymethyl-tetrahydro-pyran-2-ylmethyl)-benzaldehyde

Example 1.5.1 (1.000 g) was dissolved in dichloromethane (25 mL), andDess-Martin periodinane (1.013 g) was added. The solution was stirred 16hours at room temperature. The solution was diluted with diethyl ether(25 mL) and 2 M aqueous sodium carbonate solution (25 mL) was added. Themixture was extracted with diethyl ether three times. The organicextracts were combined, washed with brine, and dried over anhydroussodium sulfate. After filtration, the solution was concentrated underreduced pressure and purified by silica gel chromatography, eluting with50-70% ethyl acetate in heptanes. The solvent was evaporated underreduced pressure to provide the title compound. MS (ESI) me 476(M+NH₄)⁺.

1.5.3 Acetic Acid(2R,3R,4R,5S)-3,4,5-triacetoxy-6-(4-formyl-benzyl)-tetrahydro-pyran-2-ylmethylEster

Example 1.5.2 (660 mg) was dissolved in methanol (145 mL). 6 MHydrochloric acid (8 mL) was added, and the solution was stirred at roomtemperature for two days. The solvents were removed under reducedpressure, azeotroping with ethyl acetate three times. The material wasdried under vacuum for four days. The material was dissolved inN,N-dimethylformamide (50 mL). Acetic anhydride (12 mL), pyridine (6mL), and N,N-dimethylpyridin-4-amine (10 mg) were added sequentially,and the solution was stirred at room temperature for 16 hours. Thesolution was diluted with water (150 mL) and extracted with ethylacetate (50 mL) three times. The organics were combined, washed withwater, washed with brine, and dried over anhydrous sodium sulfate. Afterfiltration, the solution was concentrated under reduced pressure andpurified by chromatography on silica gel, eluting with 40-50% ethylacetate in heptanes. The solvent was evaporated under reduced pressureto provide the title compound.

1.5.4(2R,3R,4R,5S)-2-(acetoxymethyl)-6-(4-(((2-((3-((4-(6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-2-(tert-butoxycarbonyl)pyridin-3-yl)-5-methyl-1H-pyrazol-1-yl)methyl)-5,7-dimethyladamantan-1-yl)oxy)ethyl)amino)methyl)benzyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate

Example 1.5.7 (40 mg) and Example 1.5.3 (22.5 mg) were stirred indichloromethane (1 mL) at room temperature for 10 minutes. Sodiumtriacetoxyborohydride (14 mg) was added, and the solution was stirred atroom temperature for 16 hours. The material was purified bychromatography on silica gel, eluting with 10% methanol indichloromethane. The solvent was evaporated under reduced pressure toprovide the title compound. MS (ESI) me 1236 (M+H)⁺.

1.5.56-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{(2-[(4-{[(3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl]methyl}benzyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid

Example 1.5.4 (68 mg) was dissolved in methanol (0.5 mL). Aqueouslithium hydroxide solution (2M, 1 mL) was added, and the solution wasstirred at room temperature for 4.5 hours.

Acetic acid (0.1 mL) was added, and the solvents were removed undervacuum. The material was then dissolved in trifluoroacetic acid (2 mL)and stirred at room temperature for 16 hours. The solution wasconcentrated under vacuum. The residue was purified by reverse phaseHPLC using a Gilson PLC 2020 with a 150×30 mm C18 column, eluting with20-70% acetonitrile in water containing 0.1% v/v trifluoroacetic acid.The desired fractions were combined and freeze-dried to provide thetitle compound. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm 12.86 (bs,1H), 8.68 (bs, 2H), 8.04 (d, 1H), 7.80 (d, 1H), 7.62 (d, 1H), 7.51-7.43(m, 3H), 7.39-7.24 (m, 6H), 6.96 (d, 1H), 5.23 (t, 1H), 4.96 (s, 2H),4.56 (d, 1H), 4.42 (dd, 1H), 4.11 (m, 2H), 3.89 (t, 2H), 3.83 (s, 2H),3.61-3.56 (m, 3H), 3.39 (dd, 1H), 3.22 (t, 1H), 3.15 (t, 1H), 3.09 (d,1H), 3.01 (m, 6H), 2.89 (t, 1H), 2.60 (m, 1H), 2.10 (s, 3H), 1.43 (s,2H), 1.30 (q, 4H), 1.14 (m, 4H), 1.03 (q, 2H), 0.86 (s, 6H). MS (ESI)m/e 1012 (M+H)⁺.

1.6 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{(2-[(3-sulfopropyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (Compound W2.06) 1.6.13-((2-((3-((4-(6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-2-(tert-butoxycarbonyl)pyridin-3-yl)-5-methyl-1H-pyrazol-1-yl)methyl)-5,7-dimethyladamantan-1-yl)oxy)ethyl)amino)propane-1-sulfonicAcid

A mixture of Example 1.2.7 (100 mg), 1,2-oxathiolane 22-dioxide (13 mg)and N,N-diisopropylethylamine (19.07 μL) in N,N-dimethylformamide (2 mL)was heated to 50° C. overnight. The reaction was cooled and purified byreverse phase HPLC (C18 column), eluting with 20-60% acetonitrile inwater containing 0.1% v/v trifluoroacetic acid, to provide the titlecompound. MS (ESI) m/e 924.1 (M+H)⁺.

1.6.26-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(3-sulfopropyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid

Example 1.6.1 (40 mg) in dichloromethane (2.5 mL) was treated withtrifluoroacetic acid (2.5 mL) overnight. The reaction mixture wasconcentrated, and the residue was purified by reverse phasechromatography (C18 column), eluting with 20-60% acetonitrile in watercontaining 0.1% v/v trifluoroacetic acid, to provide the title compound.¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm 12.86 (s, 1H), 8.52 (s,2H), 8.04 (d, 1H), 7.79 (d, 1H), 7.61 (d, 1H), 7.41-7.55 (m, 3H),7.32-7.39 (m, 2H), 7.29 (s, 1H), 6.96 (d, 1H), 4.96 (s, 2H), 3.89 (t,2H), 3.49-3.58 (m, 2H), 2.94-3.12 (m, 6H), 2.56-2.64 (m, 2H), 1.88-1.99(m, 2H), 1.41 (s, 2H), 1.22-1.36 (m, 4H), 0.96-1.20 (m, 6H), 0.86 (s,6H). MS (ESI) m/e 868.3 (M+H)⁺.

1.7 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[(2,3-dihydroxypropyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (Compound W2.07)

To a solution of Example 1.2.7 (30 mg) in dichloromethane (3 mL) wasadded 2,3-dihydroxypropanal (3.6 mg), and NaCNBH₃ on resin (200 mg). Themixture was stirred overnight, filtered, and the solvent was evaporated.The residue was dissolved in dimethyl sulfoxide/methanol (1:1, 3 mL) andpurified by reverse phase HPLC using a Gilson system, eluting with10-85% acetonitrile in 0.1% trifluoroacetic acid in water, to give thetitle compound. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm 12.85 (s,1H), 8.27 (s, 2H), 8.03 (d, 1H), 7.79 (d, 1H), 7.61 (t, 1H), 7.33-7.54(m, 6H), 7.29 (s, 1H), 6.96 (d, 1H), 4.96 (s, 3H), 3.72-3.89 (m, 8H),3.25-3.64 (m, 6H), 2.99-3.10 (m, 4H), 2.11 (s, 3H), 1.00-1.52 (m, 8H),0.86 (s, 6H). MS (ESI) m/e 820.3 (M+H)⁺.

1.8 Synthesis of2-({[4-({[2-({3-[(4-(6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]amino}methyl)phenyl]sulfonyllamino)-2-deoxy-beta-D-glucopyranose(Compound W2.08) 1.8.1(2R,3S,4S,5R,6S)-6-(acetoxymethyl)-3-(4-formylphenylsulfonamido)tetrahydro-2H-pyran-2,4,5-triyltriacetate

4-Formylbenzene-1-sulfonyl chloride (100 mg) and(2S,3R,4R,5S,6R)-6-(acetoxymethyl)-3-aminotetrahydro-2H-pyran-2,4,5-triyltriacetate hydrochloride (563 mg) were added to 1,2-dichloroethane (4mL). N,N-Diisopropylethylamine (0.51 mL) was added, and the solution washeated at 55° C. for three days. The solution was concentrated underreduced pressure and purified by flash column chromatography on silicagel, eluting with 70% ethyl acetate in heptanes. The solvent wasevaporated under reduced pressure, and the material was dissolved inacetone (4 mL). Hydrochloric acid (1M, 4 mL) was added, and the solutionwas stirred at room temperature for 16 hours. The solution was thenextracted with 70% ethyl acetate in heptanes (20 mL). The organic layerwas washed with brine and dried over anhydrous sodium sulfate. Afterfiltration, the solvent was evaporated under reduced pressure to providethe title compound. MS (ESI) m/e 514 (M+H)⁺.

1.8.2(2R,3S,4S,5R,6S)-6-(acetoxymethyl)-3-(4-(((2-((3-((4-(6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-2-(tert-butoxycarbonyl)pyridin-3-yl)-5-methyl-1H-pyrazol-1-yl)methyl)-5,7-dimethyladamantan-1-yl)oxy)ethyl)amino)methyl)phenylsulfonamido)tetrahydro-2H-pyran-2,4,5-triylTriacetate

The title compound was prepared by substituting Example 1.8.1 forExample 1.5.3 in Example 1.5.4. MS (ESI) m/e 1301 (M+H)⁺. 1.8.32-({[4-({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]amino}methyl)phenyl]sulfonyl}amino)-2-deoxy-beta-D-glucopyranose

The title compound was prepared by substituting Example 1.8.2 forExample 1.5.4 in Example 1.5.5. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆)δ ppm 12.86 (bs, 1H), 8.87 (bs, 2H), 8.04 (d, 1H), 7.91 (d, 2H), 7.79(d, 1H), 7.70-7.55 (m, 3H), 7.52-7.42 (m, 3H), 7.39-7.33 (m, 2H), 7.29(m. 1H), 6.96 (d, 1H), 4.96 (bs, 2H), 4.85 (dd, 1H), 4.62-4.52 (m, 2H),4.32 (m, 2H), 3.89 (t, 2H), 3.83 (s, 2H), 3.70-3.35 (m, 10H), 3.02 (m,4H), 2.91 (m, 1H), 2.10 (s, 3H), 1.44 (bs, 2H), 1.37-1.22 (m, 4H),1.18-0.98 (m, 6H), 0.93-0.82 (m, 6H). MS (ESI) m/e 1075 (M+H)⁺.

1.9 Synthesis of8-(1,3-benzothiazol-2-ylcarbamoyl)-2-{6-carboxy-5-[1-({3-[2-({2-[1-(beta-D-glucopyranuronosyl)-1H-1,2,3-triazol-4-yl]ethyl}amino)ethoxy]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridin-2-yl}-1,2,3,4-tetrahydroisoquinoline(Compound W2.09) 1.9.1(2R,3R,4S,5S,6S)-2-(4-(2-hydroxyethyl)-1H-1,2,3-triazol-1-yl)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate

To a solution of(2R,3R,4S,5S,6S)-2-azido-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (720 mg) in t-butanol (8 mL) and water (4 mL) was addedbut-3-yn-1-ol (140 mg), copper(II) sulfate pentahydrate (5.0 mg) andsodium ascorbate (40 mg). The mixture was stirred 20 minutes at 100° C.under microwave conditions (Biotage Initiator). The reaction mixture wasdiluted with ethyl acetate (300 mL), washed with water and brine, anddried over sodium sulfate. Filtration and evaporation of the solventprovided the title compound. MS (ESI) m/e 430.2 (M+H)⁺.

1.9.2(2S,3S,4S,5R,6R)-2-(methoxycarbonyl)-6-(4-(2-oxoethyl)-1H-1,2,3-triazol-1-yl)tetrahydro-2H-pyran-3,4,5-triylTriacetate

To a solution of dimethyl sulfoxide (0.5 mL) in dichloromethane (10 mL)at −78° C. was added oxalyl chloride (0.2 mL). The mixture was stirred20 minutes at −78° C., and a solution of(2R,3R,4S,5S,6S)-2-(4-(2-hydroxyethyl)-1H-1,2,3-triazol-1-yl)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (233 mg) in dichloromethane (10 mL) was added through asyringe. After 20 minutes, triethylamine (1 mL) was added to themixture, and the mixture was stirred for 30 minutes while thetemperature was allowed to rise to room temperature. The reactionmixture was diluted with ethyl acetate (300 mL), washed with water andbrine, and dried over sodium sulfate. Filtration and evaporation of thesolvent gave the crude product, which was used in the next reactionwithout further purification. MS (ESI) m/e 429.2 (M+H)⁺.

1.9.38-(1,3-benzothiazol-2-ylcarbamoyl)-2-{6-carboxy-5-[1-({3-[2-({2-[1-(beta-D-glucopyranuronosyl)-1H-1,2,3-triazol-4-yl]ethyl}amino)ethoxy]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridin-2-yl}-1,2,3,4-tetrahydroisoquinoline

To a solution of Example 1.3.1 (150 mg) in dichloromethane (10 mL) wasadded Example 1.9.2 (86 mg) and NaBH₃CN on resin (2.49 mmol/g. 200 mg),and the mixture was stirred overnight. The reaction mixture was thenfiltered and concentrated. The residue was dissolved intetrahydrofuran/methanol/H₂O (2:1:1, 12 mL) and lithium hydroxidemonohydrate (50 mg) was added. The mixture was stirred overnight. Themixture was concentrated, and the residue was purified by reverse phaseHPLC using a Gilson system, eluting with 10-85% acetonitrile in 0.1%trifluoroacetic acid in water, to provide the title compound. ¹H NMR(400 MHz, dimethyl sulfoxide-d₆) δ ppm 12.84 (s, 1H), 8.48 (s, 2H), 8.20(s, 1H), 8.03 (d, 1H), 7.79 (d, 1H), 7.62 (d, 1H), 7.32-7.53 (m, 5H),7.29 (s, 1H), 6.96 (d, 1H), 5.66 (d, 1H), 4.% (s. 2H) 4.00 (d, 1H),3.76-3.92 (m, 6H), 3.22-3.26 (m, 2H), 2.96-3.15 (m, 8H), 2.10 (s, 3H),0.99-1.52 (m, 14H), 0.87 (s, 6H). MS (ESI) m/e 1028.3 (M+H)⁺.

1.10 Synthesis of3-[1-({3-[2-(2-{[4-(beta-D-allopyranosyloxy)benzyl]amino}ethoxy)ethoxy]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylicAcid (Compound W2.10) 1.10.12-(2-((3-((1H-pyrazol-1-yl)methyl)-5,7-dimethyladamantan-1-yl)oxy)ethoxy)ethanol

The title compound was prepared as in Example 1.1.4 by substitutingethane-1,2-diol with 2,2′-oxydiethanol. MS (ESI) n/e 349.2 (M+H)⁺.

1.10.22-(2-((3,5-dimethyl-7-((5-methyl-1H-pyrazol-1-yl)methyl)adamantan-1-yl)oxy)ethoxy)ethanol

The title compound was prepared as in Example 1.1.5 by substitutingExample 1.1.4 with Example 1.10.1. MS (ESI) m/e 363.3 (M+H)⁺.

1.10.32-(2-((3-((4-iodo-5-methyl-1H-pyrazol-1-yl)methyl)-5,7-dimethyladamantan-1-yl)oxy)ethoxy)ethanol

The title compound was prepared as in Example 1.1.6 by substitutingExample 1.1.5 with Example 1.10.2. MS (ESI) m/e 489.2 (M+H)⁺.

1.10.42-(2-((3-((4-iodo-5-methyl-1H-pyrazol-1-yl)methyl)-5,7-dimethyladamantan-1-yl)oxy)ethoxy)ethylMethanesulfonate

To a cooled solution of Example 1.10.3 (6.16 g) in dichloromethane (100mL) was added triethylamine (4.21 g) followed by methanesulfonylchloride (1.6 g), and the mixture was stirred at room temperature for1.5 hours. The reaction mixture was then diluted with ethyl acetate (600mL) and washed with water and brine. After drying over sodium sulfate,the solution was filtered and concentrated, and the residue was used inthe next reaction without further purification. MS (ESI) m/e 567.2(M+H)⁺.

1.10.52-(2-((3-((4-iodo-5-methyl-1H-pyrazol-1-yl)methyl)-5,7-dimethyladamantan-1-yl)oxy)ethoxy)ethanamine

A solution of Example 1.10.4 (2.5 g) in 7N ammonia in methanol (15 mL)was stirred at 100° C. for 20 minutes under microwave conditions(Biotage Initiator). The reaction mixture was concentrated under vacuum,and the residue was diluted with ethyl acetate (400 mL) and washed withaqueous NaHCO₃, water and brine. After drying over sodium sulfate, thesolution was filtered and concentrated, and the residue was used in thenext reaction without further purification. MS (EST) m/e 488.2 (M+H)⁺.

1.10.6 Tert-butyl(2-(2-((3-((4-iodo-5-methyl-1H-pyrazol-1-yl)methyl)-5,7-dimethyladamantan-1-yl)oxy)ethoxy)ethyl)carbamate

To a solution of Example 1.10.5 (2.2 g) in tetrahydrofuran (30 mL) wasadded di-ter-butyl dicarbonate (1.26 g) and 4-dimethylaminopyridine (10mg). The mixture was stirred at room temperature for 1.5 hours and wasdiluted with ethyl acetate (300 mL). The solution was washed withsaturated aqueous NaHCO₃, water (60 mL) and brine (60 mL). The organiclayer was dried with sodium sulfate, filtered and concentrated. Theresidue was purified by silica gel chromatography, eluting with 20%ethyl acetate in dichloromethane, to give the title compound. MS (ESI)me 588.2 (M+H)⁺.

1.10.7 methyl2-(6-(tert-butoxycarbonyl)-5-(1-((3-(2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)pyridin-2-yl)-1,2,3,4-tetrahydroisoquinoline-8-carboxylate

The title compound was prepared as in Example 1.2.2 by substitutingExample 1.1.6 with Example 1.10.6. MS (ESI) m/e 828.5 (M+H)⁺.

1.10.82-(6-(tert-butoxycarbonyl)-5-(1-((3-(2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)pyridin-2-yl)-1,2,3,4-tetrahydroisoquinoline-8-carboxylicAcid

The title compound was prepared as in Example 1.2.5 by substitutingExample 1.2.4 with Example 1.10.7. MS (ESI) m/e 814.5 (M+H)⁺.

1.10.10 Tert-butyl6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydrisoquinolin-2(1H)-yl)-3-(1-((3-(2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

The title compound was prepared as in Example 1.2.6 by substitutingExample 1.2.5 with Example 1.10.8. MS (ESI) m/e 946.2 (M+H)⁺.

1.10.113-(1-((3-(2-(2-aminoethoxy)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid

The title compound was prepared as in Example 1.1.17 by substitutingExample 1.1.16 with Example 1.10.9.

1.10.123-[1-({3-[2-(2-{[4-(beta-D-allopyranosyloxy)benzyl]amino}ethoxy)ethoxy]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylicacid

To a solution of Example 1.10.10 (88 mg) and triethylamine (0.04 mL) indichloromethane (1.5 mL) was added4-(((2S,3R,4R5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)benzaldehyde(27.7 mg), methanol (1 mL), MP-CNBH₃ (2.49 mmol/g, 117 mg) and aceticacid (18 μL). The reaction mixture was stirred overnight. The reactionwas filtered, and the filtrate was concentrated. The residue waspurified by purified by reverse phase chromatography (C18 column),eluting with 20-60% acetonitrile in water containing 0.1% v/vtrifluoroacetic acid, to provide the title compound. ¹H NMR (400 MHz,dimethyl sulfoxide-d₆) δ ppm 7.99 (d, 1H), 7.77 (d, 1H), 7.60 (d, 1H),7.40-7.50 (m, 2H), 7.29-7.39 (m, 6H), 6.96 (d, 2H), 6.76 (d, 1H), 5.11(d, 2H), 4.92 (s, 2H), 3.83-3.96 (m, 4H), 3.77 (s, 2H), 3.60-3.72 (m,4H), 3.01 (d, 2H), 2.80 (t, 2H), 2.09 (s, 3H), 0.98-1.32 (m, 14H), 0.82(s, 6H). MS (ESI) me 1058.3 (M+H)⁺.

1.11 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3,5-dimethyl-7-(2-{2-[(2-sulfoethyl)amino]ethoxy}ethoxy)tricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylicAcid (Compound W2.11) 1.11.1 Tert-butyl3-(1-((3-(2-(2-aminoethoxy)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinate

Example 1.10.9 (6.8 g) was dissolved in 50% trifluoroacetic acid indichloromethane (10 mL) and stirred for 20 minutes, and the solventswere removed under vacuum. The residue was purified by reverse phasechromatography, eluting with 20-80% acetonitrile in water containing0.1% trifluoroacetic acid, to provide the title compound. MS (ESI) m/e790.2 (M+H)⁺.

1.11.2 Tert-butyl6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3,5-dimethyl-7-(2-(2-((2-(phenoxysulfonyl)ethyl)amino)ethoxy)ethoxy)adamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

To a solution of Example 1.11.1 (200 mg) and N,N-diisopropylethylamine(146 μL) in tetrahydrofuran (3 mL) at 0° C. was added phenylethenesulfonate (46 mg). The reaction mixture was stirred at 0° C. for30 minutes, gradually warmed to room temperature, stirred overnight andconcentrated to provide the title compound.

1.11.36-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3,5-dimethyl-7-(2-(2-((2-(phenoxysulfonyl)ethyl)amino)ethoxy)ethoxy)adamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinicacid

A solution of Example 1.11.2 (100 mg) in dichloromethane (5 mL) wastreated with trifluoroacetic acid (2.5 mL) overnight and concentrated toprovide the title compound. MS (APCI) m/e 974.9 (M+H)⁺.

1.11.46-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(l-{[3,5-dimethyl-7-(2-{2-[(2-sulfoethyl)amino]ethoxy}ethoxy)tricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylicAcid

To a solution of Example 1.11.3 (195 mg) in tetrahydrofuran (3 mL) andmethanol (2 mL) was slowly added 1M sodium hydroxide aqueous solution (2mL). The mixture was stirred overnight, and NaOH pellets (0.5 g) wereadded. The resulting mixture was heated at 40° C. for 3 hours, cooledand concentrated. The concentrate was purified by reverse phasechromatography (C18 column), eluting with 10-70% acetonitrile in 10 mMaqueous NH₄Ac solution, to provide the title compound. ¹H NMR (400 MHz,dimethyl sulfoxide-d₆) δ ppm 8.04 (d, 1H), 7.79 (d, 1H), 7.61 (d, 1H),7.41-7.51 (m, 3H), 7.32-7.39 (m, 2H), 7.29 (s, 1H), 6.88 (d, 1H), 4.93(s, 2H), 3.89 (t, 2H), 3.81 (s, 2H), 3.60-3.66 (m, 4H), 3.13-3.19 (m,2H), 3.05-3.10 (m, 2H), 3.01 (1, 2H), 2.79 (t, 2H), 2.11 (s, 3H), 1.34(s, 2H), 1.26 (s, 4H), 0.96-1.22 (m, 6H), 0.85 (s, 6H). MS (ESI) m/e898.2 (M+H)⁺.

1.12 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-phosphonoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)-]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (Compound W2.12) 1.12.1 Tert-butyl6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-(2-((2-(diethoxyphosphoryl)ethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

To a solution of Example 1.2.7 (307 mg) in tetrahydrofuran (5 mL) wasadded diethyl vinylphosphonate (176 mg) in water (2 mL). The reactionmixture was stirred at 70° C. for 3 days, and a few drops of acetic acidwere added. The mixture was purified by reverse phase chromatography(C18 column), eluting with 10-70% acetonitrile in water containing 0.1%v/v trifluoroacetic acid, to provide the title compound. MS (APCI) m/e966.8 (M+H)⁺.

1.12.26-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-phosphonoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid

To a solution of Example 1.12.1 (170 mg) in dichloromethane (2.5 mL) wasadded bromotrimethylsilane (82 μL) and allyltrimethylsilane (50.4 μL).The reaction mixture was stirred overnight and water (0.02 mL) wasadded. The resulting mixture was stirred overnight and concentrated. Theresidue was purified by reverse phase chromatography (C18 column),eluting with 20-60% acetonitrile in water containing 0.1%trifluoroacetic acid, to provide the title compound. ¹H NMR (500 MHz,dimethyl sulfoxide-d₆) δ ppm 8.35 (s, 2H), 8.03 (d, 1H), 7.79 (d, 1H),7.62 (d, 1H), 7.41-7.53 (m, 3H), 7.33-7.40 (m, 2H), 7.29 (s, 1H), 6.96(d, 1H), 4.96 (s, 2H), 3.89 (t, 2H), 3.83 (s, 2H), 3.09 (s, 4H), 3.01(t, 2H), 2.10 (s, 3H), 1.85-2.00 (m, 2H), 1.43 (s, 2H), 1.19-1.37 (m4H), 1.14 (s, 6H), 0.87 (s, 6H). MS (APCI) m/e 854.4 (M+H)⁺.

1.13 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[methyl(3-sulfo-L-alanyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (Compound W2.13) 1.13.12-({3-[(4-iodo-5-methyl-1H-pyrazol-1-yl}methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)oxy)ethylMethanesulfonate

To a cooled solution of Example 1.1.6 (6.16 g) in dichloromethane (100mL) was added triethylamine (4.21 g) followed by methanesulfonylchloride (1.6 g), and the mixture was stirred at room temperature for1.5 hours. The reaction mixture was diluted with ethyl acetate (600 mL)and washed with water and brine. After drying over sodium sulfate, thesolution was filtered and concentrated, and the residue was used in thenext reaction without further purification. MS (ESI) m/e 523.4 (M+H)⁺.

1.13.21-({3,5-dimethyl-7-[2-(methylamino)ethoxy]tricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-4-iodo-5-methyl-1H-pyrazole

A solution of Example 1.13.1 (2.5 g) in 2M methylamine in methanol (15mL) was stirred at 100° C. for 20 minutes under microwave conditions(Biotage Initiator). The reaction mixture was concentrated under vacuum,and the residue was diluted with ethyl acetate (400 mL) and washed withaqueous NaHCO₃, water and brine. After drying over sodium sulfate, thesolution was filtered and concentrated, and the residue was used in thenext reaction without further purification. MS (ESI) m/e 458.4 (M+H)⁺.

1.13.3 Tert-butyl[2-({3-[(4-iodo-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]methylcarbamate

To a solution of Example 1.13.2 (2.2 g) in tetrahydrofuran (30 mL) wasadded di-tert-butyl dicarbonate (1.26 g) and a catalytic amount of4-dimethylaminopyridine. The mixture was stirred at room temperature for1.5 hours and diluted with ethyl acetate (300 mL). The solution waswashed with saturated aqueous NaHCO₃, water (60 mL) and brine (60 mL).The organic layer was dried with sodium sulfate, filtered andconcentrated. The residue was purified by silica gel chromatography,eluting with 20% ethyl acetate in dichloromethane, to give the titlecompound. MS (ESI) m/e 558.5 (M+H)⁺.

1.13.4 methyl2-(6-(tert-butoxycarbonyl)-5-(1-((3-(2-((tert-butoxycarbonyl)(methyl)amino)ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)pyridin-2-yl)-1,2,3,4-tetrahydroisoquinoline-8-carboxylate

To a solution of Example 1.2.1 (4.94 g) in tetrahydrofuran (60 mL) andwater (20 mL) was added Example 1.13.3 (5.57 g),1,3,5,7-tetramethyl-8-tetradecyl-2,4,6-trioxa-8-phosphaadamantane (412mg), tris(dibenzylideneacetone)dipalladium(0) (457 mg), and K₃PO₄ (11g), and the mixture was stirred at reflux for 24 hours. The reactionmixture was cooled and diluted with ethyl acetate (500 mL), washed withwater and brine. The organic layer was dried over sodium sulfate,filtered and concentrated under reduced pressure. Purification of theresidue by silica gel chromatography, eluting with 20% ethyl acetate inheptane, provided the title compound. MS (ESI) m/e 799.1 (M+H)⁺.

1.13.52-(6-(tert-butoxycarbonyl)-5-(1-((3-(2-((tert-butoxycarbonyl)(methyl)amino)ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)pyridin-2-yl)-1,2,3,4-tetrahydroisoquinoline-8-carboxylicAcid

To a solution of Example 1.13.4 (10 g) in tetrahydrofuran (60 mL),methanol (30 mL) and water (30 mL) was added lithium hydroxidemonohydrate (1.2 g), and the mixture was stirred at room temperature for24 hours. The reaction mixture was neutralized with 2% aqueous HCl andconcentrated under vacuum. The residue was diluted with ethyl acetate(800 mL) and washed with water and brine, and dried over sodium sulfate.Filtration and evaporation of the solvent provided the title compound.MS (ESI) m/e 785.1 (M+H)⁺.

1.13.6 Tert-butyl6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[(tert-butoxycarbonyl)(methyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylate

To a solution of Example 1.13.5 (10 g) in N,N-dimethylformamide (20 mL)was added benzo[d]thiazol-2-amine (3.24 g),fluoro-N,N,N′,N′-tetramethylformamidinium hexafluorophosphate (5.69 g)and N,N-diisopropylethylamine (5.57 g), and the mixture was stirred at60° C. for 3 hours. The reaction mixture was diluted with ethyl acetate(800 mL) and washed with water and brine, and dried over sodium sulfate.Filtration and evaporation of the solvent and silica gel purification ofthe residue, eluting with 20% ethyl acetate in dichloromethane, providedthe title compound. MS (ESI) m/e 915.5 (M+H)⁺.

1.13.76-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-[1-({3,5-dimethyl-7-[2-(methylamino)ethoxy]tricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylicAcid

To a solution of Example 1.13.6 (5 g) in dichloromethane (20 mL) wasadded trifluoroacetic acid (10 mL), and the mixture was stirredovernight. The solvent was evaporated under vacuum, and the residue wasdissolved in dimethyl sulfoxide/methanol (1:1, 10 mL). The mixture waspurified by reverse phase chromatography using an Analogix system and aC18 column (300 g), and eluting with 10-85% acetonitrile and 0.1%trifluoroacetic acid in water, to give the title compound.

1.13.86-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[methyl(3-sulfo-L-alanyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid

A solution of(R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-sulfopropanoic acid(0.020 g), N,N-diisopropylethylamine (0.045 mL) andO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU, 0.020 g) were stirred together inN,N-dimethylformamide (0.75 mL) at room temperature. After stirring for30 minutes, Example 1.13.7 (0.039 g) was added, and the reaction stirredfor an additional 1 hour. Diethylamine (0.027 mL) was added to thereaction and stirring was continued for 3 hours. The reaction wasdiluted with water (0.75 mL) and N,N-dimethylformamide (1 mL),neutralized with trifluoroacetic acid (0.039 mL) and purified by reversephase HPLC using a Gilson system, eluting with 20-80% acetonitrile inwater containing 0.1% v/v trifluoroacetic acid. The desired fractionswere combined and freeze-dried to provide the title compound. ¹H NMR(400 MHz, dimethyl sulfoxide-d₆) δ ppm 12.89 (s, 1H), 8.11-8.02 (m, 4H),7.84 (d, 1H), 7.66 (d, 1H), 7.60-7.45 (m, 3H), 7.45-7.36 (m, 2H), 7.34(d, 1H), 7.00 (dd. 1H), 5.00 (s, 2H), 4.57-4.40 (m, 1H), 3.93 (t, 2H),3.90-3.84 (m, 2H), 3.58-3.43 (m, 2H), 3.41-3.21 (m, 2H), 3.18-3.02 (m,3H), 2.95-2.85 (m, 2H), 2.76 (td, 2H), 2.14 (d, 3H), 1.51-0.85 (in,18H). MS (ESI) m/e 911.2 (M+H)⁺.

1.14 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(3-phosphonopropyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (Compound W2.14) 1.14.1 Di-tert-butyl (3-hydroxypropyl)phosphonate

NaH (60% in mineral oil. 400 mg) was added to di-tert-butylphosphonate(1.93 g) in N,N-dimethylformamide (30 mL), and the reaction was stirredat room temperature for 30 minutes.(3-Bromopropoxy)(tert-butyl)dimethylsilane (2.1 g) was added, and thereaction was stirred overnight. The mixture was diluted with diethylether (300 mL), and the solution was washed three times with water, andbrine, then dried over sodium sulfate, filtered, and concentrated. Theresidue was dissolved in 20 mL tetrahydrofuran, and tetrabutyl ammoniumfluoride (TBAF, 1M in tetrahydrofuran, 9 mL) was added. The solution wasstirred for 20 minutes, and then pH 7 buffer (50 mL) was added. Themixture was taken up in diethyl ethEr, and separated, and the organiclayer was washed with brine, and then concentrated. The crude productwas chromatographed on silica gel using 10-100% ethyl acetate inheptanes, followed by 5% methanol in ethyl acetate to provide the titlecompound.

1.14.2 di-Tert-butyl (3-oxopropyl)phosphonate

Example 1.14.1 (200 mg) and Dess-Martin periodinane (370 mg) werestirred in dichloromethane (5 mL) for 2 hours. The mixture was taken upin ethyl acetate, and washed twice with 1M aqueous NaOH solution, andbrine, and then concentrated. The crude product was chromatographed onsilica gel, using 50-100% ethyl acetate in heptanes followed by 10%methanol in ethyl acetate, to provide the title compound.

1.14.3 Tert-butyl6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-(2-((3-(diethoxyphosphoryl)propyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

The title compound was prepared as described in Example 1.10.11,replacing Example 1.10.10 and4-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)benzaldehydewith Example 1.2.7 and Example 1.14.2, respectively. MS (APCI) m/e 980.9(M+H)⁺.

1.14.56-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(3-phosphonopropyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid

The title compound was prepared as described in Example 1.12.2,replacing Example 1.12.1 with Example 1.14.3. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 8.37 (s, 2H), 8.03 (d. 1H), 7.79 (d, 1H), 7.62 (d,1H), 7.42-7.53 (m, 3H), 7.33-7.40 (m, 2H), 7.29 (s, 1H), 6.96 (d, 1H),4.96 (s, 2H), 3.86-3.93 (m, 2H), 3.52-3.59 (m, 2H), 2.93-3.06 (m, 6H),2.10 (s, 3H), 1.71-1.89 (m, 2H), 1.53-1.65 (m, 2H), 1.43 (s, 2H),1.23-1.37 (m, 4H), 0.%-1.19 (m, 6H), 0.87 (s, 6H). MS (APCI) m/e 868.3(M+H)⁺.

1.15 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(3-sulfo-L-alanyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (Compound W2.15)

A solution of(R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-sulfopropanoic acid(0.050 g) and O-(7-azabenaotriazol-1-yl)-N,N,N′N′-tetramethyluroniumhexafluorophosphate (0.049 g) were dissolved in N,N-dimethylformamide (1mL) and N,N-diisopropylethylamine (0.102 mL) was added. After stirringfor 15 minutes, Example 1.3.1 (0.100 g) was added, and the reactionstirred for an additional 3 hours. Diethylamine (0.061 mL) was added tothe reaction and stirring was continued overnight. The reaction wasneutralized with 2,2,2-trifluoroacetic acid (0.090 mL) and diluted withN,N-dimethylfornamide (1 mL) and water (1 mL). The mixture was purifiedby reverse phase HPLC using a Gilson system, eluting with 20-80%acetonitrile in water containing 0.1% v/v trifluoroacetic acid. Thedesired fractions were combined and freeze-dried to provide the titlecompound. ¹H NMR (500 MHz, dimethyl sulfoxide-d₆) δ ppm 12.86 (s, 1H),8.63 (t, 1H), 8.15-8.01 (m, 4H), 7.79 (d, 1H), 7.62 (d, 1H), 7.56-7.41(m, 3H), 7.40-7.33 (m, 2H), 7.30 (s, 1H), 6.96 (d, 1H), 4.96 (s, 2H),4.08-3.97 (m, 1H), 3.89 (t, 2H), 3.82 (s, 2H), 3.42-3.31 (m, 2H),3.28-3.17 (m, 1H), 3.16-3.06 (m, 1H), 3.01 (t, 2H), 2.97 (dd, 1H), 2.76(dd, 1H), 2.10 (s, 3H), 1.39 (s, 2H), 1.32-1.20 (m, 4H), 1.19-1.07 (m,4H), 1.07-0.95 (m, 2H), 0.85 (s, 6H). MS (ESI) m/e 897.2 (M+H)⁺.

1.16 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3,5-dimethyl-7-(2-{2-[(3-phosphonopropyl)amino]ethoxy}ethoxy)tricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylicAcid (Compound W2.16) 1.16.1 Tert-butyl6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-(2-(2-((3-(di-tert-butoxyphosphoryl)propyl)amino)ethoxy)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

Example 1.10.10 (338 mg) and Example 1.14.2 (120 mg) were dissolved inethanol (20 mL), and the solution was concentrated. The residue wasagain taken up in ethanol (20 mL) and concentrated. The residue was thendissolved in dichloromethane (10 mL) and to this was added sodiumtriacetoxyborohydride (119 mg), and the reaction was stirred overnight.The crude mixture was chromatographed on silica gel, using 1%triethylamine in 95:5 ethyl acetate/methanol, to provide the titlecompound. MS (ESI) 1080.3 (M+H)⁺.

1.16.26-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3,5-dimethyl-7-(2-{2-[(3-phosphonopropyl)amino]ethoxy}ethoxy)tricyclo[3.3.1.17]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylicAcid

Example 1.16.1 (22 mg) was stirred in dichloromethane (3 mL) andtrifluoroacetic acid (3 mL) for 2 days. The mixture was concentrated andchromatographed via reverse phase on a Biotage Isolera One system usinga 40 g C18 column and eluting with 10-90/acetonitrile in 0.1%trifluoroacetic acid/water, to provide the title compound as atrifluoroacetic acid salt. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm□8.62 (bs, 1H), 8.10 (d, 1H), 7.86 (d, 1H), 7.68 (d, 1H), 7.57 (d, 1H),7.54 (dd, 1H), 7.50 (d, 1H), 7.42 (m, 2H), 7.35 (s, 1H), 7.02 (d, 1H),5.02 (s, 2H), 3.94 (m, 2H), 3.97 (m, 2H), 3.68 (m, 2H), 3.55 (m, 2H),3.15 (m, 1H), 3.09 (m, 4H), 2.55 (m, 4H), 2.15 (s, 3H), 1.86 (m, 1H),1.66 (m, 2H), 1.45 (m, 2H), 1.31 (m, 4H), 1.19 (m, 4H), 1.08 (m, 2H),0.90 (s, 6H). MS (ESI) 912.2 (M+H)⁺.

1.17 Synthesis of3-{1-[(3-{2-[L-alpha-aspartyl(methyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylicAcid (Compound W2.17) 1.17.16-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1,H)-yl]-3-{1-[(3-{2-[{(2S)-4-tert-butoxy-2-[(tert-butoxycarbonyl)amino]-4-oxobutanoyl}(methyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid

A solution of Example 1.13.7 (0.060 g), (S)-4-tert-butyl1-(2,5-dioxopyrrolidin-1-yl) 2-((tert-butoxycarbonyl)amino)succinate(0.034 g) and N,N-diisopropylethylamine were stirred together indichloromethane (1 mL). After stirring overnight, the reaction wasloaded onto silica gel and eluted using a gradient of 0.5-5%methanol/dichloromethane to give the title compound.

1.17.23-{1-[(3-{2-[L-alpha-aspartyl(methyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylicAcid

A solution of Example 1.17.1 (0.049 g) in dichloromethane (1 mL) wastreated with trifluoroacetic acid (0.5 mL), and the reaction was stirredovernight. The reaction was concentrated, dissolved inN,N-dimethylformamide (2 mL) and water (0.5 mL) then purified by reversephase HPLC using a Gilson system, eluting with 20-80% acetonitrile inwater containing 0.1% v/v trifluoroacetic acid. The desired fractionswere combined and freeze-dried to provide the title compound. ¹H NMR(400 MHz, dimethyl sulfoxide-d₆) δ ppm 12.85 (s, 1H), 8.15 (d, 3H), 8.03(d, 1H), 7.79 (d, 1H), 7.62 (d, 1H), 7.55-7.41 (m, 3H), 7.36 (td. 2H),7.29 (d, 1H), 6.95 (d, 1H), 4.96 (s, 2H), 4.55 (s, 1H), 3.92-3.86 (m,2H), 3.60-3.47 (m, 2H), 3.47-3.37 (m, 2H), 3.32-3.21 (m, 1H), 3.09-2.97(m, 4H), 2.92-2.72 (m, 3H), 2.67-2.53 (m, 1H), 2.10 (s, 3H), 1.46-0.94(m, 12H), 0.85 (s, 6H). MS (ESI) m/e 875.2 (M+H)⁺.

1.18 Synthesis of6-{4-[({2-[2-(2-aminoethoxy)ethoxy]ethyl}[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]amino)methyl]benzyl}-2,6-anhydro-L-gulonicAcid (Compound W2.18) 1.18.1(2S,3S,4R,5S)-3,4,5-Triacetoxy-6-(4-bromomethyl-benzyl)-tetrahydro-pyran-2-carboxylicAcid Methyl Ester

The title compound was prepared as described in J. R. Walker et al.,Bioorg. Med. Chem. 2006,14, 3038-3048. MS (ESI) m/e 518, 520 (M+NH₄)⁺.

1.18.2(2S,3S,4R,5S)-3,4,5-Triacetoxy-6-(4-formyl-benzyl)-tetrahydro-pyran-2-carboxylicAcid Methyl Ester

Example 1.18.1 (75 mg) and pyridine N-oxide (14 mg) were added toacetonitrile (0.75 mL). Silver (I) oxide (24 mg) was added to thesolution, and the solution was stirred at room temperature for 16 hours.Anhydrous sodium sulfate (5 mg) was added, and the solution was stirredfor five minutes. The solution was filtered and concentrated. The crudematerial was purified by flash column chromatography on silica gel,eluting with 50-70% ethyl acetate in heptanes. The solvent wasevaporated under reduced pressure to provide the title compound.

1.18.3(3R,4S,5R,6R)-2-(4-(((2-((3-((4-(6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-2-(tert-butoxycarbonyl)pyridin-3-yl)-5-methyl-1H-pyrazol-1-yl)methyl)-5,7-dimethyladamantan-1-yl)oxy)ethyl)amino)methyl)benzyl)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate

The title compound was prepared by substituting Example 1.18.2 forExample 1.5.3 in Example 1.5.4. MS (ESI) m/e 1222 (M+H)⁺.

1.18.4 {2-[2-(2-Oxo-ethoxy)-ethoxy]-ethyl}-carbamic Acid Tert-ButylEster

The title compound was prepared by substituting{2-[2-(2-hydroxy-ethoxy)-ethoxy]-ethyl}-carbamic acid tert-butyl esterfor Example 1.5.1 in Example 1.5.2.

1.18.5(3R4S,R,6R)-2-(4-(2-(2-((3-((4-(6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-34-dihydroisoquinolin-2(1H)-yl)-2-(tert-butoxycarbonyl)pyridin-3-yl)-5-methyl-1H-pyrazol-1-yl)methyl)-5,7-dimethyladamantan-1-yl)oxy)ethyl)-14,14-dimethyl-12-oxo-5,8,13-trioxa-2,11-diazapentadecyl)benzyl)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate

The title compound was prepared by substituting Example 1.18.3 forExample 1.2.7 and Example 1.18.4 for Example 1.5.3 in Example 1.5.4. MS(ESI) m/e 1453 (M+H)⁺.

1.18.66-{4-[({2-[2-(2-aminoethoxy)ethoxy]ethyl}[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]amino)methyl]benzyl}-2,6-anhydro-L-gulonicAcid

The title compound was prepared by substituting Example 1.18.5 forExample 1.5.4 in Example 1.5.5. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆)δ ppm 9.38 (bs, 1H), 8.05 (dd, 1H), 7.90-7.68 (m, 6H), 7.62 (m, 2H),7.53-7.27 (m, 8H), 6.94 (d, 1H), 4.96 (bs, 1H), 4.38 (bs, 4H), 3.91-3.57(m, 11H), 3.37-3.11 (m, 14H), 2.98 (m, 6H), 2.61 (m, 1H), 2.10 (s, 3H),1.44 (bs, 2H), 1.26 (m, 4H), 1.18-0.90 (m, 6H), 0.87 (bs, 6H). MS (ESI)m/e 1157 (M+H)⁺.

1.19 Synthesis of4-({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]amino}methyl)phenylhexopyranosiduronic Acid (Compound W2.19) 1.19.1(2R,3S,4R,5R,6R)-2-(4-formylphenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate

To a solution of(2R,3R,4S,5S,6S)-2-bromo-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (2.42 g) in acetonitrile (30 mL) was added silver(I) oxide(1.4 g) and 4-hydroxybenzaldehyde (620 mg). The reaction mixture wasstirred for 4 hours and filtered. The filtrate was concentrated, and theresidue was purified by silica gel chromatography, eluting with 5-50%ethyl acetate in heptanes, to provide the title compound. MS (ESI) m/e439.2 (M+H)⁺.

1.19.24-({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]amino}methyl)phenylhexopyranosiduronic acid

To a solution of Example 1.2.7 (36 mg) in tetrahydrofuran (2 mL) andacetic acid (0.2 mL) was added Example 1.19.1 (21 mg) followed by MgSO₄(60 mg). The mixture was stirred for 1 hour before the addition ofNaBH₃CN on resin (153 mg). The mixture was then stirred for 3 hours. Themixture was filtered and lithium hydroxide monohydrate (20 mg) was addedto the filtrate. The mixture was stirred for 2 hours and was acidifiedwith trifluoroacetic acid and purified by reverse phase HPLC (Gilsonsystem), eluting with 10-85% acetonitrile in 0.1% trifluoroacetic acidin water, to give the title compound. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 12.86 (s, 1H), 8.57-8.72 (m, 2H), 8.03 (d, 1H), 7.79(d, 1H), 7.62 (d, 1H), 7.34-7.53 (m, 6H), 7.08 (t, 2H), 6.95 (d, 1H),5.10 (d, 1H), 4.96 (s, 2H), 4.06-4.15 (m, 4H), 3.83-3.97 (m, 6H),3.26-3.42 (m, 8H), 2.93-3.10 (m, 6H), 2.10 (s, 3H), 1.43 (s, 2H),1.24-1.38 (m, 6H), 0.97-1.16 (m, 4H), 0.86 (s, 6H). MS (ESI) m/e 1028.3(M+H)⁺.

1.20 Synthesis of6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-phosphonoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)-]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (Compound W2.20) 1.20.12-((3,5-dimethyl-7-((5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)methyl)adamantan-1-yl)oxy)ethanol

To a solution of Example 1.1.6 (9 g) and[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II)dichloromethane (827 mg) in acetonitrile (60 mL) was added triethylamine(10 mL) and pinacolborane (6 mL). The mixture was stirred at refluxovernight, cooled and used directly in the next step. MS (ESI) m/e 445.4(M+H)⁺.

1.20.2 Tert-buty6-chloro-3-(1-((3-(2-hydroxyethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

To a solution of tert-butyl 3-bromo-6-chloropicolinate (5.92 g) intetrahydrofuran (60 mL) and water (30 mL) was added the crude Example1.20.1 (4.44 g),1,3,5,7-tetramethyl-6-phenyl-2,4,8-trioxa-6-phosphaadamante (1.5 g),tris(dibenzylideneacetone)dipalladium(0) (927 mg) and K₃PO₄(22 g). Themixture was stirred at reflux overnight, cooled, diluted with ethylacetate (800 mL) and washed with water and brine. The organic layer wasdried over sodium sulfate, filtered and concentrated. The residue waspurified by flash chromatography, eluting with 20% ethyl acetate inheptane followed by 5% methanol in dichloromethane, to give the titlecompound. MS (ESI) m/e 531.1 (M+H)⁺.

1.20.3 Tert-butyl3-(1-((3-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-chloropicolinate

To a solution of Example 1.20.2 (3.2 g) in N,N-dimethylformamide (20 mL)was added imidazole (0.62 g) and chloro t-butyldimethylsilane (1.37 g).The mixture was stirred overnight, diluted with ethyl acetate (300 mL),and washed with water and brine. The organic layer was dried over sodiumsulfate, filtered, and concentrated. The residue was purified by flashchromatography, eluting with 20% ethyl acetate in heptanes, to providethe title compound. MS (ESI) m/e 645.4 (M+H)⁺.

1.20.4 Tert-butyl3-(1-((3-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(1,2,3,4-tetrahydroquinolin-7-yl)picolinate

To a solution of7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,2,3,4-tetrahydroquinoline(507 mg) in 1,4-dioxane (10 mL) and water (5 mL) was added Example1.20.3 (1.25 g), bis(triphenylphosphine)palladium(II)dichloride (136mg), and cesium fluoride (884 mg). The mixture was heated at 120° C. ina microwave synthesizer (Biotage, Initiator) for 20 minutes. The mixturewas diluted with ethyl acetate (500 mL), and washed with water andbrine. The organic layer was dried over sodium sulfate, filtered,concentrated and purified by flash chromatography, eluting with 20%ethyl acetate in heptanes and then with 5% methanol in dichloromethane,to provide the title compound. MS (ESI) m/e 741.5 (M+H)⁺.

1.20.5 Tert-butyl6-(1-(benzo[d]thiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl)-3-(1-(3-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

To a suspension of bis(2,5-dioxopyrrolidin-1-yl) carbonate (295 mg) inacetonitrile (10 mL) was added benzo[d]thiazol-2-amine (173 mg), and themixture was stirred for 1 hour. A solution of Example 1.20.4 (710 mg) inacetonitrile (10 mL) was added, and the suspension was stirredovernight. The mixture was diluted with ethyl acetate (300 mL), washedwith water and brine and dried over sodium sulfate. After filtration,the organic layer was concentrated and purified by silica gelchromatography, eluting with 20% ethyl acetate in heptanes, to providethe title compound. MS (ESI) m/e 917.2 (M+H)⁺.

1.20.6 Tert-butyl6-(1-(benzo[d]thiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl)-3-(1-((3-(2-hydroxyethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

To a solution of Example 1.20.5 (1.4 g) in tetrahydrofuran (10 mL) wasadded tetrabutyl ammonium fluoride (1.0 M in tetrahydrofuran, 6 mL). Themixture was stirred for 3 hours, diluted with ethyl acetate (300 mL) andwashed with water and brine. The organic layer was dried over sodiumsulfate, filtered, and concentrated to provide the title compound. MS(ESI)/e 803.4 (M+H)⁺.

1.20.7 Tert-butyl6-(1-(benzo[d]thiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl)-3-(1-((3,5-dimethyl-7-(2-((methylsulfonyl)oxy)ethoxy)adamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

To a cooled (0° C.) solution of Example 1.20.6 (1.2 g) indichloromethane (20 mL) and triethylamine (2 mL) was addedmethanesulfonyl chloride (300 mg). The mixture was stirred for 4 hours,diluted with ethyl acetate (200 mL) and washed with water and brine. Theorganic layer was dried over sodium sulfate, filtered, and concentratedto provide the title compound. MS (ESI) me 881.3 (M+H)⁺.

1.20.8 Tert-butyl3-(1-((3-(2-azidoethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(1-(benzo[d]thiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl)picolinate

To a solution of Example 1.20.7 (1.5 g) in N,N-dimethylformamide (20 mL)was added sodium azide (331 mg). The mixture was stirred for 48 hours,diluted with ethyl acetate (20.0 mL) and washed with water and brine.The organic layer was dried over sodium sulfate, filtered, concentratedand purified by silica gel chromatography, eluting with 20% ethylacetate in dichloromethane, to provide the title compound. MS (ESI) m/e828.4 (M+H)⁺.

1.20.9 Tert-butyl3-(1-((3-(2-aminoethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(1-(benzo[d]thiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl)picolinate

To a solution of Example 1.20.8 (1.5 g) in tetrahydrofuran (30 mL) wasadded Pd/C (10%. 200 mg). The mixture was stirred under a hydrogenatmosphere overnight. The reaction was filtered, and the filtrate wasconcentrated to provide the title compound. MS (ESI) m/e 802.4 (M+H)⁺.

1.20.10 Tert-butyl6-(1-(benzo[d]thiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl)-3-(1-((3-(2-((2-(diethoxyphosphoryl)ethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

The title compound was prepared as described in Example 1.12.1,replacing Example 1.2.7 with Example 1.20.9.

1.20.116-[1-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-phosphonoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid

The title compound was prepared as described in Example 1.12.2,replacing Example 1.12.1 with Example 1.20.10. ¹H NMR (500 MHz, dimethylsulfoxide-d₆) a ppm 8.40 (s, 2H), 8.02 (d, 1H), 7.74-7.89 (m, 3H), 7.47(s, 2H), 7.38 (t, 1H), 7.30 (d, 1H), 7.23 (t, 1H), 3.96 (s, 2H), 3.90(s, 2H), 3.53-3.64 (m, 2H), 3.03-3.18 (m, 2H), 2.84 (t, 2H), 2.23 (s,3H), 1.87-2.02 (m, 4H), 1.46 (s, 2H), 1.26-1.38 (m, 4H), 1.12-1.23 (m,4H), 0.99-1.11 (m, 2H), 0.89 (s, 6H). MS (ESI) m/e 854.1 (M+H)⁺.

1.21 Synthesis of6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]-3-{1-[(3,5-dimethyl-7-{2-[methyl(3-sulfo-L-alanyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (Compound W2.21) 1.21.1 Tert-butyl(2-((3,5-dimethyl-7-((5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)methyl)adamantan-1-yl)oxy)ethyl)(methyl)carbamate

To a solution of Example 1.13.3 (1.2 g) in 1,4-dioxane was addedbis(benzonitrile)palladium(II) chloride (0.04 g).4,4,5,5-tetramethyl-1,3,2-dioxaborolane (0.937 mL) and triethylamine(0.9 mL). The mixture was heated at reflux overnight, diluted with ethylacetate and washed with water (60 mL) and brine (60 mL). The organiclayer was dried over sodium sulfate, filtered and concentrated toprovide the title compound.

1.21.2 Tert-butyl3-(1-((3-(2-((tert-butoxycarbonyl)(methyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-chloropicolinate

The title compound was prepared as described in Example 1.1.12,replacing Example 1.1.11 and Example 1.1.8 with tert-butyl3-bromo-6-chloropicolinate and Example 1.21.1, respectively. MS (APCI)m/e 643.9 (M+H)⁺.

1.21.3 Tert-butyl3-(1-((3-(2-((tert-butoxycarbonyl)(methyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(1,2,3,4-tetrahydroquinolin-7-yl)picolinate

A mixture of Example 1.21.2 (480 mg).7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,2,3,4-tetrahydroquinoline(387 mg), dichlorobis(triphenylphosphine)-palladium(II) (78 mg) andcesium fluoride (340 mg) in 1,4-dioxane (12 mL) and water (5 mL) washeated at 100° C. for 5 hours. The reaction was cooled and diluted withethyl acetate. The resulting mixture was washed with water and brine,and the organic layer was dried over sodium sulfate, filtered, andconcentrated. The residue was purified by flash chromatography, elutingwith 50% ethyl acetate in heptanes, to provide the title compound. MS(APCI) m/e 740.4 (M+H)⁺.

1.21.4 Tert-butyl6-(1-(benzo[d]thiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl)-3-(1-((3-(2-((tert-butoxycarbonyl)(methyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

To a solution of benzo[d]thiazol-2-amine (114 mg) in acetonitrile (5 mL)was added bis(2,5-dioxopyrrolidin-1-yl) carbonate (194 mg). The mixturewas stirred for 1 hour, and Example 1.21.3 (432 mg) in acetonitrile (5mL) was added. The mixture was stirred overnight, diluted with ethylacetate, washed with water and brine. The organic layer was dried oversodium sulfate, filtered, and concentrated. The residue was purified bysilica gel chromatography, eluting with 50% ethyl acetate in heptanes,to provide the title compound.

1.21.56-(1-(benzo[d]thiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl)-3-(1-((3,5-dimethyl-7-(2-(methylamino)ethoxy)adamantan-1-yl)methy)-5-methyl-1H-pyrazol-4-yl)picolinicAcid

Example 1.2.4 (200 mg) in dichloromethane (5 mL) was treated withtrifluoroacetic acid (2.5 mL) overnight. The mixture was concentrated toprovide the title compound. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δppm 8.40 (s, 1H), 8.30 (s, 2H), 8.02 (d, 1H), 7.85 (d, 1H), 7.74-7.83(m, 2H), 7.42-7.53 (m, 2H), 7.38 (t, 1H), 7.30 (d, 1H), 7.23 (t, 1H),3.93-4.05 (m, 2H), 3.52-3.62 (m, 2H), 2.97-3.10 (m, 2H), 2.84 (t, 2H),2.56 (t, 2H), 2.23 (s, 3H), 1.88-2.00 (m, 2H), 1.45 (s, 2H), 1.25-1.39(m, 4H), 1.12-1.22 (m, 4H), 1.00-1.09 (m, 2H), 0.89 (s, 6H). MS (ESI) me760.1 (M+H)⁺.

1.21.66-(1-(benzo[d]thiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl)-3-(1-((3-(2-((R)-2-((tert-butoxycarbonyl)amino)-N-methyl-3-sulfopropanamido)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinicacid

(R)-2-((tert-butoxycarbonyl)amino)-3-sulfopropanoic acid (70.9 mg) andO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU, 65 mg) in N,N-dimethylformamide (1.5 ml) wascooled in ice-bath, and N,N-diisopropylethylamine (68.9 μL) was added.The mixture was stirred at 0° C. for 15 minutes and at room temperaturefor 8 hours. Example 1.21.5 (100 mg) in N,N-dimethylformamide (1 mL) andN,N-diisopropylethylamine (60 μL) were added. The resulting mixture wasstirred overnight, concentrated and purified by reverse phasechromatography (C18 column), eluting with 20-60% acetonitrile in watercontaining 0.1% trifluoroacetic acid, to provide the title compound.

1.21.76-[1-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]-3-{1-[(3,5-dimethyl-7-{2-[methyl(3-sulfo-L-alanyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid

Example 1.21.6 (80 mg) in dichloromethane (3 mL) was treated withtrifluoroacetic acid (1.5 mL) for 20 minutes. The reaction mixture wasconcentrated and purified by reverse phase chromatography (C18 column),eluting with 0-50% acetonitrile in 4 mM aqueous ammonium acetatesolution, to provide the title compound. ¹H NMR (500 MHz dimethylsulfoxide-d₆) δ ppm 8.57 (s, 1H), 7.59-7.67 (m, 3H), 7.54 (d, 1H),7.46-7.51 (m, 1H), 7.30 (d, 1H), 7.08-7.17 (m, 2H), 6.90 (t, 1H),3.91-4.10 (m, 3H), 3.84 (s, 2H), 3.04 (s, 2H), 2.75-2.83 (m, 4H),2.59-2.70 (m, 2H), 2.27-2.39 (m, 2H), 2.26 (s, 3H), 1.81-1.93 (m, 2H),1.74 (s, 9H), 1.42 (s, 2H), 0.96-1.33 (m, 10H), 0.86 (s, 3H). MS (ESI)me 909.2 (M−H)⁻.

1.22 Synthesis of3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-([1,3]thiazolo[5,4-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylicAcid (Compound W2.22) 1.22.1 Tert-butyl3-(1-((3-(2-azidoethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(thiazolo[5,4-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinate

Example 1.2.5 (560 mg) and thiazolo[5,4-b]pyridin-2-amine (135 mg) weredissolved in dichloromethane (12 mL). N,N-Dimethylpyridin-4-amine (165mg) and N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride(260 mg) were added, and the reaction stirred at room temperatureovernight. The reaction mixture was concentrated, and the crude residuewas purified by silica gel chromatography, eluting with 6535dichloromethane/ethyl acetate, to provide the title compound. MS (ESI)m/e 829.1 (M+H)⁺.

1.22.2 Tert-butyl3-(1-((3-(2-aminoethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(thiazolo[5,4-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinate

The title compound was prepared by substituting Example 1.22.1 forExample 1.2.6 in Example 1.2.7. MS (ESI) m/e 803.2 (M+H)⁺.

1.22.3 Tert-butyl3-[1-({3,5-dimethyl-7-[(2,2,7,7-tetramethyl-10,10-dioxido-3,3-diphenyl-4,9-dioxa-10λ⁶-thia-13-aza-3-silapentadecan-15-yl)oxy]tricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]-6-[8-([1,3]thiazolo[5,4-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylate

To a solution of Example 1.22.2 (70 mg) and4-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylbutyl ethenesulfonate (48mg) in dichloromethane (1 mL) was added N,N-diisopropylethylamine (0.06mL), and the reaction stirred at room temperature overnight. Thereaction was concentrated, and the crude residue was purified by silicagel chromatography, eluting with a gradient of 1-4% methanol indichloromethane, to provide the title compound. MS (ESI) m/e 1249.2(M+H)⁺.

1.22.42-((2-((3-((4-(2-(tert-butoxycarbonyl)-6-(8-(thiazolo[5,4-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)pyridin-3-yl)-5-methyl-1H-pyrazol-1-yl)methyl)-5,7-dimethyladamantan-1-yl)oxy)ethyl)amino)ethanesulfonicAcid

To a solution of Example 1.22.3 (70 mg) in tetrahydrofuran (0.25 mL) wasadded tetrabutylammonium fluoride (60 μL, 1.0 M solution intetrahydrofuran), and the reaction was stirred at room temperature fortwo days. The reaction was concentrated, and the residue was purified byreverse phase chromatography (C18 column), eluting with 10-90%acetonitrile in water containing 0.1% trifluoroacetic acid, to providethe title compound as a trifluoroacetic acid salt. MS (ESI) m/e 911.1(M+H)⁺.

1.22.53-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-([1,3]thiazolo[5,4-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylicAcid

The title compound was prepared by substituting Example 1.22.4 forExample 1.2.8 in Example 1.2.9. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆)δ ppm 13.00 (s, 1H), 8.52 (dd, 2H), 8.33 (br s, 2H), 8.16 (dd, 1H), 7.62(m, 1H), 7.53 (m, 2H), 7.45 (d, 1H), 7.38 (m, 1H), 7.29 (s, 1H), 6.98(d, 1H), 4.96 (s, 2H), 3.88 (m, 2H), 3.83 (s, 2H), 3.54 (m, 2H), 3.22(m, 2H), 3.10 (m, 2H), 3.02 (t, 2H), 2.80 (t, 2H), 2.11 (s, 3H), 1.41(s, 2H), 1.28 (m, 4H), 1.14 (m, 4H), 1.02 (m, 2H), 0.86 (s, 6H). MS(ESI) me 855.2 (M+H)⁺.

1.23 Synthesis of3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-([1,3]thiazolo[4,5-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylicAcid (Compound W2.23) 1.23.1 Tert-butyl3-(1-((3-(2-azidoethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(thiazolo[4,5-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinate

The title compound was prepared by substitutingthiazolo[4,5-b]pyridin-2-amine for thiazolo[5,4-b]pyridin-2-amine inExample 1.22.1. MS (ESI) m/e 855.2 (M+H)⁺.

1.23.2 Tert-butyl3-(1-((3-(2-aminoethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(thiazolo[4,5-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinate

The title compound was prepared by substituting Example 1.23.1 forExample 1.2.6 in Example 1.2.7. MS (ESI) m/e 803.2 (M+H)⁺.

1.23.3 Tert-butyl3-[1-({3,5-dimethyl-7-[(2,2,7,7-tetramethyl-10,10-dioxido-3,3-diphenyl-4,9-dioxa-10λ⁶-thia-13-aza-3-silapentadecan-15-yl)oxy]tricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]-6-[8-([1,3]thiazolo[4,5-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylate

The title compound was prepared by substituting Example 1.23.2 forExample 1.22.2 in Example 1.22.3. MS (ESI) m/e 1249.2 (M+H)⁺.

1.23.43-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-([1,3]thiazolo[4,5-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylicAcid

The title compound was prepared by substituting Example 1.23.3 forExample 1.2.8 in Example 1.2.9. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆)δ ppm 13.20 (br s, 1H), 8.61 (dd, 1H), 8.56 (dd, 1H), 8.33 (br s, 2H),7.56 (d, 1H) 7.52 (d, 1H), 7.46 (d, 1H), 7.39 (m, 2H), 7.29 (s, 1H),6.98 (d, 1H), 4.98 (s, 2H), 3.88 (m, 2H), 3.83 (s, 2H), 3.54 (m, 2H),3.22 (m, 2H), 3.10 (m, 2H), 3.02 (t. 2H), 2.80 (t, 2H), 2.10 (s, 3H),1.41 (s, 2H), 1.30 (m, 4H), 1.12 (m, 4H), 1.02 (m, 2H), 0.86 (s, 6H). MS(ESI) m/e 855.1 (M+H)⁺.

1.24 Synthesis of6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (Compound W2.24) 1.24.1 Tert-butyl6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]-3-[1-({3,5-dimethyl-7-[(2,2,7,7-tetramethyl-10,10-dioxido-3,3-diphenyl-4,9-dioxa-10λ⁶-thia-13-aza-3-silapentadecan-15-yl)oxy]tricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylate

The title compound was prepared as described in Example 1.2.8, replacingExample 1.2.7 with Example 1.20.9.

1.24.26-[1-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid

The title compound was prepared as described in Example 1.2.9, replacingExample 1.2.8 with Example 1.24.1. ¹H NMR (500 MHz, dimethylsulfoxide-d₆) δ ppm 8.26-8.46 (m, 3H), 8.02 (d, 1H), 7.89 (d, 1H), 7.82(d, 1H), 7.75-7.79 (m, 1H), 7.47 (s, 2H), 7.37 (t, 1H), 7.30 (d, 1H),7.22 (t, 1H), 3.96 (s, 2H), 3.90 (s, 2H), 3.54-3.61 (m, 2H), 3.18-3.29(m, 2H), 3.07-3.15 (m, 2H), 2.78-2.92 (m, 4H), 2.23 (s, 3H), 1.87-2.02(m, 2H), 1.44 (s, 2H), 1.32 (q, 4H), 1.12-1.25 (m, 4H), 1.00-1.11 (m,2H), 0.88 (s, 6H). MS (ESI) m/e 854.0 (M+H)⁺.

1.25 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[(2-carboxyethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (W2.25) 1.25.1 Tert-butyl6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-(2-((3-(tert-butoxy)-3-oxopropyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

The title compound was prepared as described in Example 1.12.1,replacing diethyl vinylphosphonate with tert-butyl acrylate. MS (APCI)me 930.6 (M+H)⁺.

1.25.26-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[(2-carboxyethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid

The title compound was prepared as described in Example 1.6.2, replacingExample 1.6.1 with Example 1.25.1. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 8.03 (d, 1H), 7.78 (d, 1H), 7.61 (d, 1H), 7.39-7.50(m, 2H), 7.32-7.38 (m, 3H), 7.23 (s, 1H), 6.73 (d, 1H), 4.88 (s, 2H),3.88 (t. 2H), 3.79 (s, 2H), 2.99 (t, 2H), 2.86-2.93 (m, 2H), 2.50-2.58(m, 2H), 2.08 (s, 3H), 1.35 (d, 2H), 1.01-1.30 (m, 10H), 0.86 (s, 6H).MS (APCI) me 819.0 (M+H)⁺.

1.26 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(3-phosphonopropyl)piperidin-4-yl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicacid (Compound W2.26) 1.26.1 Tert-butyl6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-(((1r,3r)-3-(2-((1-(tert-butoxycarbonyl)piperidin-4-yl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

A solution of Example 1.2.7 (0.020 g), tert-butyl4-oxopiperidine-1-carboxylate (4.79 mg) and sodium triacetoxyborohydride(7 mg) was stirred in dichloromethane (0.5 mL) at room temperature. Thereaction was stirred overnight and purified without workup by silica gelchromatography, eluting with 0 to 10% methanol in dichloromethane, togive the title compound. MS (ELSD) m/e 985.4 (M+H)⁺.

1.26.26-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(3-phosphonopropyl)(piperidin-4-yl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid

A solution of Example 1.26.1 (0.108 g), Example 1.14.2 (0.030 g) andsodium triacetoxyborohydride (0.035 g) in dichloromethane (1 mL) wasstirred at room temperature for 1 hour. Trifluoroacetic acid (1 mL) wasadded to the reaction, and stirring was continued overnight. Thereaction was concentrated, dissolved in N,N-dimethylformamide (2 mL) andwater (0.5 mL) and purified by reverse phase HPLC using a Gilson system,eluting with 10-75% acetonitrile in water containing 0.1% v/vtrifluoroacetic acid. The desired fractions were combined andfreeze-dried to provide the title compound. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 8.83 (s, 1H), 8.50 (s. 1H), 8.04 (d, 2H), 7.80 (d,2H), 7.63 (d, 2H), 7.56-7.42 (m, 5H), 7.37 (tt, 3H), 7.30 (s, 1H), 6.%(d, 1H), 4.% (s, 21), 3.89 (t, 2H), 3.44 (d, 6H), 3.31-3.16 (m, 6H),3.09-2.98 (m, 2H), 2.98-2.85 (m, I1H), 2.18 (d, 2H), 2.10 (s, 3H),2.00-1.74 (m, 4H), 1.71-1.57 (m, 2H), 1.51-0.97 (m, 12H), 0.87 (s, 6H).MS (ESI) m/e 951.2 (M+H)⁺.

1.27 Synthesis of3-{1-[(3-{2-[D-alpha-aspartyl(methyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylicAcid (Compound W2.27) 1.27.1 Tert-butyl6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3,5-dimethyl-7-(2-(methylamino)ethoxy)adamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

The title compound was prepared as described in Example 1.11.1 bysubstituting Example 1.10.9 with Example 1.13.6.

1.27.23-{1-[(3-{2-[D-alpha-aspartyl(methyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylicAcid

A solution of Example 1.27.1 (0.074 g),2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisouroniumhexafluorophosphate(V) (0.038 g), N,N-diisopropylethylamine (0.048 mL)and (R)-4-(tert-butoxy)-2-((tert-butoxycarbonyl)amino)-4-oxobutanoicacid (0.029 g) in dichloromethane (1 mL) was stirred for 2 hours.Trifluoroacetic acid (0.5 mL) was added, and stirring was continuedovernight. The reaction was concentrated, dissolved inN,N-dimethylformamide (1.5 mL) and water (0.5 mL), and purified byreverse phase HPLC using a Gilson system, eluting with 10-75%acetonitrile in water containing 0.1% v/v trifluoroacetic acid. Thedesired fractions were combined and freeze-dried to provide the titlecompound. ¹H NMR (500 MHz, dimethyl sulfoxide-d₆) δ ppm 12.88 (s, 1H),8.16 (s, 3H), 8.04 (d, 1H), 7.80 (d, 1H), 7.62 (d, 1H), 7.55-7.42 (m,3H), 7.41-7.33 (m, 2H), 7.33-7.27 (m, 1H), 6.96 (d, 1H), 4.96 (s, 2H),4.63-4.49 (m, 1H), 3.89 (t, 2H), 3.82 (s. 2H), 3.61-3.37 (m, 4H),3.10-2.97 (m, 4H), 2.89-2.73 (m, 2H), 2.67-2.52 (m, 1H), 2.10 (s, 3H),1.45-0.95 (m, 12H), 0.85 (s, 6H). MS (ESI) m/e 875.3 (M+H)⁺.

1.28 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-({[3-(2-({[1-(carboxymethyl)piperidin-4-yl]amino}ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)-]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxyliacid (Compound W2.28)

A solution of Example 1.2.7 (0.055 g), tert-butyl2-(4-oxopiperidin-1-yl)acetate (0.014 g) and sodiumtriacetoxyborohydride (0.019 g) was stirred in dichloromethane (0.5 mL)at room temperature. After stirring for 2 hours, trifluoroacetic acid(0.5 mL) was added to the reaction, and stirring was continuedovernight. The reaction was concentrated, dissolved inN,N-dimethylformamide (1.5 mL) and water (0.5 mL) and purified byreverse phase HPLC using a Gilson system, eluting with 10-80%acetonitrile in water containing 0.1% v/v trifluoroacetic acid. Thedesired fractions were combined and freeze-dried to provide the titlecompound. ¹H NMR (501 MHz, dimethyl sulfoxide-d₆) δ ppm 12.85 (s, 1H),8.80 (s, 2H), 8.03 (d, 1H), 7.80 (d, 1H), 7.62 (d, 1H), 7.55-7.41 (m,3H), 7.36 (q, 2H), 7.29 (s, 1H), 6.96 (d, 1H), 4.96 (s, 2H), 4.07 (s,2H), 3.89 (t, 2H), 3.83 (s, 2H), 3.66-3.55 (m, 4H), 3.30 (s, 1H), 3.08(s, 4H), 3.02 (t, 2H), 2.22 (d, 2H), 2.10 (s, 3H), 1.97-1.78 (m, 2H),1.44 (s, 2H), 1.31 (q, 4H), 1.20-0.96 (m, 6H), 0.87 (s, 6H). MS (ESI)m/e 887.3 (M+H)⁺.

1.29 Synthesis ofN-[(5S)-5-amino-6-{[2-({3-[(4-(6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl)-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](methyl)amino}-6-oxohexyl]-N,N-dimethylmethanaminium(Compound W2.29)

A solution of Fmoc-N-ε-(trimethyl)-L-lysine hydrochloride (0.032 g),2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisouroniumhexafluorophosphate(V) (0.028 g) and N,N-diisopropylethylamine (0.034mL) in N,N-dimethylformamide (0.5 mL) was stirred for 5 minutes. Thereaction was added to Example 1.13.7 (0.050 g), and stirring wascontinued at room temperature overnight. Diethylamine (0.069 mL) wasadded to the reaction, and stirring was continued for an additional 2hours. The reaction was diluted with N,N-dimethylformamide (1 mL), water(0.5 mL), and trifluoroacetic acid (0.101 mL). The mixture was purifiedby reverse phase HPLC using a Gilson system, eluting with 10-90%acetonitrile in water containing 0.1% v/v trifluoroacetic acid. Thedesired fractions were combined and freeze-dried to provide the titlecompound. ¹H NMR (500 MHz, dimethyl sulfoxide-d₆) δ ppm 12.87 (s, 1H),8.13 (s, 3H), 8.04 (d, 1H), 7.80 (d, 1H), 7.62 (d, 1H), 7.54-7.42 (m,3H), 7.42-7.34 (m, 2H), 7.29 (s, 1H), 6.96 (d, 1H), 4.96 (s, 2H),4.42-4.24 (m, 1H), 3.89 (t, 2H), 3.82 (s, 2H), 3.29-3.16 (m, 2H),3.08-3.00 (m, 15H), 2.87 (s, 2H), 2.10 (s, 3f), 1.84-1.60 (m, 4H),1.42-0.97 (m, 15H), 0.85 (s, 6H). MS (ESI) m/e 930.3 (M+H)⁺.

1.30 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[piperidin-4-yl(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (Compound W2.30) 1.30.1 Tert-butyl6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3-({13-[1-(tert-butoxycarbonyl)piperidin-4-yl]-2,2,7,7-tetramethyl-10,10-dioxido-3,3-diphenyl-4,9-dioxa-10λ⁶-thia-13-aza-3-silapentadecan-15-yl}oxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylate

A solution of Example 1.2.8 (0.111 g), tert-butyl4-oxopiperidine-1-carboxylate (0.021 g) and sodium triacetoxyborohydride(0.028 g) in dichloromethane (1 mL) was stirred at room temperature for1 hour. Acetic acid (7.63 μL) was added, and stirring was continuedovernight. Additional tert-butyl 4-oxopiperidine-1-carboxylate (0.021g), sodium triacetoxyborohydride (0.028 g) and acetic acid (8 μL) wereadded to the reaction, and stirring was continued for an additional 4hours. The reaction was loaded directly onto silica gel and eluted witha gradient of 0.5-4% methanol in dichloromethane to give the titlecompound.

1.30.26-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[piperidin-4-yl(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid

To a solution of Example 1.30.1 (0.078 g) in dichloromethane (1 mL) wasadded trifluoroacetic acid (0.5 mL), and the reaction was stirred atroom temperature overnight. The reaction was concentrated and dissolvedin N,N-dimethylformamide (1.5 mL) and water (0.5 mL). The mixture waspurified by reverse phase HPLC using a Gilson system, eluting with10-75% acetonitrile in water containing 0.1% v/v trifluoroacetic acid.The desired fractions were combined and freeze-dried to provide thetitle compound. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm 12.89 (s,1H), 9.31 (s, 1H), 8.75 (d, 1H), 8.36-8.19 (m, 1H), 8.08 (d, 1H), 7.84(d, 1H), 7.66 (d, 1H), 7.58 (d, 1H), 7.55-7.45 (m, 2H), 7.40 (td, 2H),7.34 (s, 1H), 6.99 (d, 1H), 5.00 (s, 2H), 3.93 (t, 2H), 3.87 (s, 2H),3.49 (d, 6H), 3.39-3.31 (m, 2H), 3.01 (m, 6H), 2.15 (s, 6H), 1.94 (s,2H), 1.58-0.99 (m, 12H), 0.91 (s, 6H). MS (ESI) m/e 937.3 (M+H)⁺.

1.31 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-5-(3-phosphonopropoxy)-3,4-dihydroisoquinolin-2(1H)-yl]-3-[1-({3,5-dimethyl-7-[2-(methylamino)ethoxy]tricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylicAcid (Compound W2.31) 1.31.1 Tert-butyl8-bromo-5-hydroxy-3,4-dihydroisoquinoline-2(1H)-carboxylate

To a solution of tert-butyl5-hydroxy-3,4-dihydroisoquinoline-2(1H)-carboxylate (9 g) inN,N-dimethylformamide (150 mL) was added N-bromosuccinimide (6.43 g).The mixture was stirred overnight and quenched with water (200 mL). Themixture was diluted with ethyl acetate (500 mL), washed with water andbrine, and dried over sodium sulfate. Evaporation of the solvent gavethe title compound, which was used in the next reaction without furtherpurification. MS(ESI) m/e 329.2 (M+H)⁺.

1.31.2 Tert-butyl5-(benzyloxy)-8-bromo-3,4-dihydroisoquinoline-2(1H)-carboxylate

To a solution of Example 1.31.1 (11.8 g) in acetone (200 mL) was addedbenzyl bromide (7.42 g) and K₂CO₃ (5 g), and the mixture was stirred atreflux overnight. The mixture was concentrated, and the residue waspartitioned between ethyl acetate (600 mL) and water (200 mL). Theorganic layer was washed with water and brine, dried over sodiumsulfate, filtered and concentrated. The residue was purified by silicagel chromatography, eluting with 10% ethyl acetate in heptane, toprovide the title compound. MS (ESI) m/e 418.1 (M+H)⁺.

1.31.3 2-tert-butyl 8-methyl5-(benzyloxy)-3,4-dihydroisoquinoline-2,8(1H)-dicarboxylate

Methanol (100 mL) and triethylamine (9.15 mL) were added to Example1.31.2 (10.8 g) and[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (0.48 g) ina 500 mL stainless steel pressure reactor. The vessel was sparged withargon several times. The reactor was pressurized with carbon monoxideand stirred for 2 hours at 100° C. under 60 psi of carbon monoxide.After cooling, the crude reaction mixture was concentrated under vacuum.The residue was added to ethyl acetate (500 mL) and water (200 mL). Theorganic layer was further washed with water and brine, dried over sodiumsulfate, filtered and concentrated. The residue was purified by silicagel chromatography, eluting with 10-20% ethyl acetate in heptane, toprovide the title compound. MS (ESI) m/e 398.1 (M+H)⁺.

1.31.4 Methyl 5-(benzyloxy)-1,2,3,4-tetrahydroisoquinoline-8-carboxylateHydrochloride

To a solution of Example 1.31.3 (3.78 g) in tetrahydrofuran (20 mL) wasadded 4N HCl in 1,4-dioxane (20 mL), and the mixture was stirredovernight. The mixture was concentrated under vacuum to give the titlecompound, which was used in the next reaction without furtherpurification. MS(ESI) m/e 298.1 (M+H)⁺.

1.31.5 Methyl5-(benzyloxy)-2-(5-bromo-6-(tert-butoxycarbonyl)pyridin-2-yl)-1,2,3,4-tetrahydroisoquinoline-8-carboxylate

To a solution of Example 1.31.4 (3.03 g) in dimethyl sulfoxide (50 mL)was added Example 1.1.10 (2.52 g) and triethylamine (3.8 mL), and themixture was stirred at 60° C. overnight under nitrogen. The reactionmixture was diluted with ethyl acetate (500 mL), washed with water andbrine, dried over sodium sulfate, filtered and concentrated. The residuewas purified by silica gel chromatography, eluting with 20% ethylacetate in heptane, to give the title compound. MS (ESI) m/e 553.1(M+H)⁺.

1.31.6 Tert-butyl(2-((3,5-dimethyl-7-((5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)methyl)adamantan-1-yl)oxy)ethyl)(methyl)carbamate

To a solution of Example 1.13.3 (2.6 g) and[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II)dichloromethane (190 mg) in acetonitrile (30 mL) was added triethylamine(2.0 mL) and pinacolborane (1.4 mL), and the mixture was stirred atreflux overnight. The mixture was used directly in the next reactionwithout work up. MS (ESI) m/c 558.4 (M+H)⁺.

1.31.7 methyl5-(benzyloxy)-2-(6-(tert-butoxycarbonyl)-5-(1-((3-(2-((tert-butoxycarbonyl)(methyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)pyridin-2-yl)-1,2,3,4-tetrahydroisoquinoline-8-carboxylate

To a solution of Example 1.31.5 (2.58 g) in tetrahydrofuran (40 mL) andwater (20 mL) was added Example 1.31.6 (2.66 g),1,3,5,7-tetramethyl-6-phenyl-2,4,8-trioxa-6-phosphaadamante (341 mg),tris(dibenzylideneacetone)dipalladium(0) (214 mg), and K₃PO₄(4.95 g),and the mixture was stirred at reflux for 4 hours. The mixture wasdiluted with ethyl acetate (500 mL), washed with water and brine, driedover sodium sulfate, filtered and concentrated. The residue was purifiedby silica gel chromatography, eluting with 20% ethyl acetate indichloromethane, to provide the title compound. MS (ESI) m/e 904.5(M+H)⁺.

1.31.8 methyl2-(6-(tert-butoxycarbonyl)-5-(1-((3-(2-((tert-butoxycarbonyl)(methyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)pyridin-2-yl)-5-hydroxy-1,2,3,4-tetrahydroisoquinoline-8-carboxylate

Example 1.31.7 (3.0 g) in tetrahydrofuran (60 mL) was added to Pd(OH)₂(0.6 g, Degussa # E101NE/W, 20% on carbon, 49% water content) in a 250mL stainless steel pressure bottle. The mixture was shaken for 16 hoursunder 30 psi of hydrogen gas at 50° C. The mixture was filtered througha nylon membrane, and the solvent was evaporated under vacuum to providethe title compound. MS (ESI) m/e 815.1 (M+H)⁺.

1.31.9 methyl2-(6-(tert-butoxycarbonyl)-5-(1-((3-(2-((tert-butoxycarbonyl)(methyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)pyridin-2-yl)-5-(3-(di-tert-butoxyphosphoryl)propoxy)-1,2,3,4-tetrahydroisoquinoline-8-carboxylate

To a solution of Example 1.31.8 (163 mg) in tetrahydrofuran (10 mL) wasadded Example 1.14.1 (50.5 mg), triphenylphosphine (52.5 mg) anddi-tert-butylazodicarboxylate (46.2 mg), and the mixture was stirred for3 hours. The mixture was diluted with ethyl acetate (200 mL), washedwith water and brine, dried over sodium sulfate, filtered andconcentrated. The residue was purified by silica gel chromatography,eluting with 20% ethyl acetate in heptanes followed by 5% methanol indichloromethane, to provide the title compound. MS (ESI) m/e 1049.2(M+H)⁺.

1.31.102-(6-(tert-butoxycarbonyl)-5-(1-((3-(2-((tert-butoxycarbonyl)(methyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)pyridin-2-yl)-5-(3-(di-tert-butoxyphosphoryl)propoxy)-1,2,3,4-tetrahydroisoquinoline-8-carboxylicAcid

To a solution of Example 1.31.9 (3 g) in tetrahydrofuran (20 mL),methanol (10 mL) and water (10 mL) was added lithium hydroxidemonohydrate (30 mg), and the mixture was stirred at room temperature for24 hours. The reaction mixture was neutralized with 2% aqueous HCl andconcentrated under vacuum. The residue was diluted with ethyl acetate(800 mL), washed with water and brine, and dried over sodium sulfate.Filtration and evaporation of solvent provided the title compound. MS(ESI) m/e 1034.5 (M+H)⁺.

1.31.116-[8-(1,3-benzothiazol-2-ylcarbamoyl)-5-(3-phosphonopropoxy)-3,4-dihydroisoquinolin-2(1H)-yl]-3-[1-({3,5-dimethyl-7-[2-(methylamino)ethoxy]tricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylicAcid

To a solution of Example 1.31.10 (207 mg) in N,N-dimethylformamide (4mL) was added benzo[d]thiazol-2-amine (45.1 mg, 0.3 mmol),fluoro-N,N,N′,N′-tetramethylformamidinium hexafluorophosphate (79 mg)and N,N-diisopropylethylamine (150 mg), and the mixture was stirred at60° C. for 3 hours. The reaction mixture was diluted with ethyl acetate(200 mL,) washed with water and brine, dried over sodium sulfate,filtered and concentrated. The residue was purified by silica gelchromatography, eluting with 20% ethyl acetate in heptane followed by 5%methanol in dichloromethane. After concentration, the material wasdissolved in a mixture of dichloromethane and trifluoroacetic acid (1:1,6 mL) and was allowed to sit at room temperature overnight. The solventwas evaporated, and the residue was dissolved in dimethylsulfoxide/methanol (1:1, 9 mL).

The mixture was purified by reverse phase HPLC using a Gilson system,eluting with 10-85% acetonitrile in water containing 0.1% v/vtrifluoroacetic acid, to give the title compound. ¹H NMR (501 MHz,dimethyl sulfoxide-d₆) δ ppm 8.27 (s, 2H), 8.02 (d, 1H), 7.76 (dd, 2H),7.43-7.56 (m, 2H), 7.32-7.37 (m, 1H), 7.29 (s, 1H), 7.00 (dd, 2H), 5.02(s, 2H), 4.15 (t, 2H), 3.88-3.93 (m, 2H), 3.83 (s. 3f). 3.50-3.59 (m,4H), 2.95-3.08 (m, 2H), 2.78-2.87 (m, 2H), 2.51-2.55 (m, 3H), 2.11 (s,3H), 1.90-2.01 (m, 2H), 1.65-1.75 (m, 2H), 1.41 (s, 2H), 1.22-1.36 (m,6H), 0.98-1.18 (m, 6H), 0.87 (s, 6H). MS (ESI)/e 898.2 (M+H)⁺.

1.32 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3-(2-{[N-(2-carboxyethyl)-L-alpha-aspartyl]amino}ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylicAcid (Compound W2.32) 1.32.1 Tert-butyl6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-(2-((S)-4-(tert-butoxy)-2-((tert-butoxycarbonyl)amino)-4-oxobutanamido)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

To a cold (0° C.) solution of(S)-4-(tert-butoxy)-2-((tert-butoxycarbonyl)amino)-4-oxobutanoic acid(136 mg) and O-(7-azabenotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU, 179 mg) in N,N-dimethylformamide (3 mL) wasadded N,N-diisopropylethylamine (165 μL). The reaction mixture wasstirred for 10 minutes, and Example 1.2.7 (252 mg) inN,N-dimethylformamide (1 mL) was added. The mixture was stirred at roomtemperature for 1.5 hours and was purified by reverse phasechromatography (C18 column), eluting with 50-100% acetonitrile in watercontaining 0.1% v/v trifluoroacetic acid, to provide the title compound.

1.32.23-(1-((3-(2-((S)-2-amino-3-carboxypropanamido)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid

Example 1.32.1 (100 mg) in dichloromethane (3 mL) was treated withtrifluoroacetic acid (2.5 mL) overnight. The reaction mixture wasconcentrated to provide the title compound.

1.32.36-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-(2-((S)-2-((3-(tert-butoxy)-3-oxopropyl)amino)-3-carboxypropanamido)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinicAcid

To a mixture of Example 1.32.2 (102 mg) and N,N-diisopropylethylamine(0.21 mL) in N,N-dimethylformamide (1.5 mL) was added tert-butylacrylate (80 mg) and water (1.5 mL). The mixture was heated at 50° C.for 24 hours and purified by reverse phase chromatography (C18 column),eluting with 20-60% acetonitrile in water containing 0.1% v/vtrifluoroacetic acid, to provide the title compound. MS (APCI) m/e 989.1(M+H)⁺.

1.32.46-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3-(2-{[N-(2-carboxyethyl)-L-alpha-aspartyl]amino}ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylicAcid

The title compound was prepared as described in Example 1.6.2, replacingExample 1.6.1 with Example 1.32.3. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 12.86 (s, 3H), 8.62-9.21 (m, 2H), 8.52 (t, 1H), 8.03(d, 1H), 7.79 (d, 1H), 7.62 (d, 1H), 7.42-7.53 (m, 3H), 7.33-7.41 (m,2H), 7.29 (s, 1H), 6.95 (d, 1H), 4.96 (s, 2H), 4.04-4.19 (m, 1H), 3.89(t, 2H), 3.81 (s, 2H), 3.32-3.41 (m, 2H), 3.16-3.27 (m, 2H), 3.10 (t,2H), 3.01 (t, 2H), 2.83 (d, 2H), 2.66 (t, 2H), 2.10 (s, 3H), 1.39 (s,2H), 1.20-1.32 (m, 4H), 0.94-1.16 (m, 6H), 0.85 (s, 6H). MS (ESI) m/e933.2 (M+H)⁺.

1.33 Synthesis of3-{1-[(3-{2-[(2-aminoethyl)(2-sulfoethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylicAcid (Compound W2.33) 1.33.16-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-(2-((2-((tert-butoxycarbonyl)amino)ethyl)(2-sulfoethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinicAcid

To a solution of Example 1.2.9 (188 mg), tert-butyl(2-oxoethyl)carbamate (70.1 mg) and N,N-diisopropylethylamine (384 μL)was added sodium triacetoxyborohydride (140 mg), and the mixture wasstirred overnight. NaCNBH₃ (13.83 mg) was added. The resulting mixturewas stirred for 1 hour, and methanol (1 mL) was added. The mixture wasstirred for 10 minutes, diluted with ethyl acetate, and washed withbrine. The organic layer was dried over sodium sulfate, filtered andconcentrated. The residue was purified by reverse phase chromatography(C18 column), eluting with 20-80% acetonitrile in water containing 0.1%v/v trifluoroacetic acid, to provide the title compound.

1.33.23-{1-[(3-{2-[(2-aminoethyl)(2-sulfoethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylicAcid

The title compound was prepared as described in Example 1.6.2, replacingExample 1.6.1 with Example 1.33.1. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 12.85 (s, 1H), 8.03 (d, 1H), 7.87 (s, 2H), 7.79 (d,1H), 7.62 (d, 1H), 7.41-7.56 (m, 3H), 7.33-7.40 (m, 2H), 7.29 (s, 1H),6.% (d, 1H), 4.% (s. 2H), 3.89 (t, 2H), 3.50 (s, 2H), 3.29-3.40 (m, 4H),3.19 (s, 2H), 3.01 (t, 2H), 2.94 (t, 2H), 2.11 (s, 3H), 1.43 (s, 2H),1.25-1.37 (m, 4H), 0.98-1.19 (m, 6H), 0.87 (s, 6H). MS (ESI) m/e 897.2(M+H)⁺.

1.34 Synthesis of6-[5-(2-aminoethoxy)-8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-[1-({3,5-dimethyl-7-[2-(methylamino)ethoxy]tricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylicAcid (Compound W2.34) 1.34.1 methyl5-(2-(((benzyloxy)carbonyl)amino)ethoxy)-2-(6-(tert-butoxycarbonyl)-5-(1-((3-(2-((tert-butoxycarbonyl)(methyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)pyridin-2-yl)-1,2,3,4-tetrahydroisoquinoline-8-carboxylate

To a mixture of Example 1.31.8 (500 mg), benzyl(2-hydroxyethyl)carbamate (180 mg) and triphenyl phosphine (242 mg) intetrahydrofuran (9 mL) was added (E)-di-tert-butydiazene-1,2-dicarboxylate (212 mg). The mixture was stirred for 2 hours,diluted with ethyl acetate and washed with water and brine. The organiclayer was dried over sodium sulfate, filtered, and concentrated. Theresidue was purified by silica gel chromatography, eluting with 50-100%ethyl acetate in heptanes, to provide the title compound. MS (APCI) m/e991.1 (M+H)⁺.

1.34.25-(2-(((benzyloxy)carbonyl)amino)ethoxy)-2-(6-(tert-butoxycarbonyl)-5-(1-((3-(2-((tert-butoxycarbonyl)(methyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)pyridin-2-yl)-1,2,3,4-tetrahydroisoquinoline-8-carboxylicacid

To a solution of Example 1.34.1 (480 mg) in tetrahydrofuran (10 mL) andmethanol (5 mL) was added 1 M lithium hydroxide (1.94 mL). The mixturewas heated at 50° C. overnight, cooled, acidified with 10% aqueous HClto pH 3 and concentrated. The residue was purified by reverse phasechromatography (C18 column), eluting with 40-99/o acetonitrile in watercontaining 0.1% v/v trifluoroacetic acid, to provide the title compound.MS (ESI) m/e 977.4 (M+H)⁺.

1.34.3 Tert-butyl6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-5-(2-(((benzyloxy)carbonyl)amino)ethoxy)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-(2-((tert-butoxycarbonyl)(methyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

To a mixture of Example 1.34.2 (245 mg), benzo[d]thiazol-2-amine (151mg) and fluoro-N,N,N′,N′-tetramethylformamidinium hexafluorophosphate(TFFH) (132 mg) in N,N-dimethylformamide (3 mL) was addedN,N-diisopropylethylamine (876 μL). The reaction mixture was heated at65° C. for 24 hours, cooled, diluted with ethyl acetate and washed withwater and brine.

The organic layer was dried over sodium sulfate, filtered andconcentrated. The residue was purified by silica gel chromatography,eluting with 0-80% ethyl acetate in heptanes, to provide the titlecompound. MS (APCI) m/e 1109.5 (M+H)⁺.

1.34.46-[5-(2-aminoethoxy)-8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-[1-({3,5-dimethyl-7-[2-(methylamino)ethoxy]tricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylicacid

Example 1.34.3 (100 mg) in dichloromethane (0.5 mL) was treated withtrifluoroacetic acid (10 mL) overnight. The reaction mixture wasconcentrated and purified by reverse phase chromatography (C18 column),eluting with 20-60% acetonitrile in water containing 0.1% v/vtrifluoroacetic acid, to provide the title compound. ¹H NMR (400 MHz,dimethyl sulfoxide-d₆) δ ppm 12.75 (s, 2H), 8.27 (s, 2H), 7.89-8.09 (m,4H), 7.77 (s, 2H), 7.44-7.53 (m, 2H), 7.35 (t, 1H), 7.29 (s, 1H), 7.02(dd, 2H), 5.02 (s, 2H), 4.27 (t, 2H), 3.87-3.97 (m, 2H), 3.83 (s, 2H),3.50-3.58 (m, 2H), 3.00 (s, 2H), 2.88-2.96 (m, 2H), 2.52-2.60 (m, 2H),2.10 (s, 3H), 1.42 (s, 2H), 1.23-1.36 (m, 4H), 0.98-1.19 (m, 6H), 0.87(s, 6H). MS (ESI) m/e 819.3 (M+H)⁺.

1.35 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-3-{1-[(3,5-dimethyl-7-{2-[(3-sulfopropyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (Compound W2.35) 1.35.1 Tert-butyl6-chloro-3-(1-((3,5-dimethyl-7-(2-oxoethoxy)adamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

To a solution of oxalyl chloride (8 mL, 2.0 M in dichloromethane) indichloromethane (20 mL) at −78° C., was added dropwise dimethylsulfoxide (1 mL) in dichloromethane (10 mL) over 20 minutes. Thesolution was stirred for 30 minutes under argon, and Example 1.20.2 (3.8g) as a solution in dichloromethane (30 mL) was added over 10 minutes.The reaction mixture was stirred at −78° C. for an additional 60minutes. Triethylamine (2 mL) was added at −78° C., and the reactionmixture was stirred for 60 minutes. The cooling bath was removed, andthe reaction allowed to warm to room temperature overnight. Water (60mL) was added. The aqueous layer was acidified with 1% aqueous HClsolution and extracted with dichloromethane. The combined organic layerswere washed with 1% aqueous HCl solution, aqueous NaHCO₃ solution, andbrine. The organic layer was dried over sodium sulfate and concentratedto provide the title compound. MS (ESI) m/e 527.9 (M+H)⁺.

1.35.2 2,2,2-trifluoro-1-(p-tolyl)ethyl 3-iodopropane-1-sulfonate

The title compound was prepared according to a procedure reported in J.Org. Chem., 2013, 78, 711-716.

1.35.3 2,2,2-trifluoro-1-(p-tolyl)ethyl 3-aminopropane-1-sulfonate

A solution of Example 1.35.2 (2.0 g) in 7 N ammonia in methanol (20 mL)was heated to 80° C. under microwave conditions (Biotage Initiator) for45 minutes. The mixture was concentrated, and the residue was dissolvedin ethyl acetate (300 mL). The organic layer was washed with water andbrine, dried over sodium sulfate, filtered, and concentrated to providethe title compound. MS (ESI) m/e 312.23 (M+H)⁺.

135.4 Tert-butyl6-chloro-3-(1-(((3,5-dimethyl-7-(2-((3-((2,2,2-trifluoro-1-(p-tolyl)ethoxy)sulfonyl)propyl)amino)ethoxy)adamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

To a solution of Example 1.35.3 (1.96 g) in dichloroethane (30 mL) wasadded Example 1.35.1 (3.33 g). The reaction mixture was stirred at roomtemperature for 1 hour, and a suspension of NaBH₄ (1.2 g) in methanol (8mL) was added. The mixture was stirred at room temperature for 3 hoursand diluted with ethyl acetate (300 mL). The organic layer was washedwith 2N aqueous NaOH, water, and brine, dried over sodium sulfate,filtered and concentrated. The residue was dissolved in tetrahydrofuran(30 mL), and di-tert-butyl dicarbonate (2 g) was added followed by theaddition of catalytic amount of 4-dimethylaminopyridine. The mixture wasstirred at room temperature overnight. The mixture was diluted withethyl acetate (300 mL) and washed with water and brine. The organiclayer was dried over sodium sulfate filtered, and concentrated toprovide the title compound. MS (ESI) m/e 924.42 (M+H)⁺.

1.35.57-(6-(tert-butoxycarbonyl)-5-(1-((3-(2-((tert-butoxycarbonyl)(3-((2,2,2-trifluoro-1-(p-tolyl)ethoxy)sulfonyl)propyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)pyridin-2-yl)-1-naphthoicAcid

To a solution of methyl7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-naphthoate (203 mg) ina mixture of 1,4-dioxane (10 mL) and water (5 mL) was added Example1.35.4 (600 mg), bis(triphenylphosphine)palladium(II)dichloride (45.6mg), and cesium fluoride (296 mg). The mixture was heated at 120° C.under microwave conditions (Biotage Initiator) for 30 minutes, dilutedwith ethyl acetate (200 mL), and washed with water and brine. Theorganic layer was dried over sodium sulfate, filtered, and concentrated.The residue was purified by silica gel chromatography, eluting with 20%ethyl acetate in heptane, to provide an ester intermediate. The residuewas dissolved in a mixture of tetrahydrofuran (8 mL), methanol (4 mL)and water (4 mL), and was treated with lithium hydroxide monohydrate(200 mg) for 3 hours. The reaction was acidified with 1N aqueous HCl topH 4 and was diluted with ethyl acetate (400 mL). The resulting mixturewas washed with water and brine. The organic layer was dried over sodiumsulfate, filtered, and concentrated to provide the title compound. MS(ESI) m/e 1060.24 (M+H)⁺.

1.35.66-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-3-{1-[(3,5-dimethyl-7-{2-[(3-sulfopropyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-y)methy]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid

To a solution of Example 1.35.5 (405 mg) in dichloromethane (10 mL) wasadded benzo[d]thiazol-2-amine (57.4 mg),1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide hydrochloride (146 mg)and 4-(dimethylamino)pyridine (93 mg). The mixture was stirred at roomtemperature overnight, diluted with ethyl acetate (200 mL), and washedwith water and brine. The organic layer was dried over sodium sulfate,filtered, and concentrated. The residue was dissolved in dichloromethane(3 mL) and treated with trifluoroacetic acid (3 mL) overnight. Thereaction mixture was concentrated, and the residue was purified byreverse phase HPLC (Gilson system), eluting with a gradient of 10-85%acetonitrile in water containing 0.1% v/v trifluoroacetic acid, toprovide the title compound. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δppm 13.08 (s, 1H), 9.00 (s, 1H), 8.53 (s, 2H), 8.36 (dd, 1H), 8.26-8.13(m, 3H), 8.06 (dd, 1H), 8.04-7.97 (m, 1H), 7.94 (d, 1H), 7.80 (d, 1H),7.69 (dd, 1H), 7.51-7.43 (m, 2H), 7.40-7.31 (in, 1H), 7.19 (d, OH), 3.88(s, 2H), 3.54 (t, 2H), 3.16-2.91 (m, 4H), 2.68-2.55 (m, 2H), 2.29 (s,0H), 2.22 (s, 3H), 1.93 (p, 2H), 1.43 (s, 2H), 1.38-1.23 (m, 4H), 1.10(dq, 6H), 0.87 (s, 6H). MS (ESI) m/e 863.2 (M+H)⁺.

1.36 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[(2-carboxyethyl)(piperidin-4-yl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (Compound W2.36) 1.36.1 Tert-butyl6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-(((1r,3r)-3-(2-((3-(tert-butoxy)-3-oxopropyl)(1-(tert-butoxycarbonyl)piperidin-4-yl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

A solution of Example 1.25.1 (0.086 g), tert-butyl4-oxopiperidine-1-carboxylate (0.037 g), sodium triacetoxyborohydride(0.039 g) and acetic acid (11 μL) in dichloromethane (1 mL) was stirredat room temperature. After stirring overnight, the reaction was loadedonto silica gel and eluted using a gradient of 0.5 to 5% methanol indichloromethane to give the title compound. MS (ELSD) m/e 1113.5 (M+H)⁺.

1.36.26-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[(2-carboxyethyl)(piperidin-4-yl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid

A solution of Example 1.36.1 (0.050) in dichloromethane (0.5 mL) wastreated with trifluoroacetic acid (0.5 mL), and the reaction was stirredovernight. The reaction was concentrated and dissolved in dimethylsulfoxide and methanol (1:1). The mixture was purified by reverse phaseHPLC using a Gilson system, eluting with 10-75% acetonitrile in watercontaining 0.1% v/v trifluoroacetic acid. The desired fractions werecombined and freeze-dried to provide the title compound. ¹H NMR (400MHz, dimethyl sulfoxide-d₆) δ ppm 12.84 (s, 1H), 9.38 (s, 1H), 8.78 (s,1H), 8.42 (s, 1H), 8.03 (d, 1H), 7.80 (d, 1H), 7.63 (d, 1H), 7.55-7.42(m, 3H), 7.41-7.33 (m, 2H), 7.30 (s, 1H), 6.96 (d, 1H), 4.96 (s, 2H),3.89 (t, 2H), 3.83 (s, 2H), 3.73-3.54 (m, 3H), 3.53-3.34 (m, 4H).3.34-3.25 (m, 2H), 3.02 (t, 2H), 2.99-2.85 (m, 2H), 2.78 (t, 2H),2.23-2.04 (m, 5H), 1.92-1.76 (m, 2H), 1.43 (s, 2H), 1.39-1.23 (m, 4H),1.23-0.96 (m, 6H), 0.87 (s, 6H). MS (ESI) m/e 901.3 (M+H)⁺.

1.37 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(3-sulfo-L-alanyl)(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (Compound W2.37)

A solution of(R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-sulfopropanoic acid(0.011 g) and2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisouroniumhexafluorophosphate(V) (10.80 mg) in N,N-dimethylformamide (0.5 mL) wasstirred for 5 minutes. This solution was added to Example 1.2.9 (0.025g) and N,N-diisopropylethylamine (0.014 mL). After stirring for 2 hours,diethylamine (0.013 mL) was added to the reaction, and stirring wascontinued for an additional 1 hour. The reaction was diluted withN,N-dimethylformamide and water and quenched with trifluoroacetic acid.The mixture was purified by reverse phase HPLC using a Gilson system,eluting with 10-75% acetonitrile in water containing 0.1% v/vtrifluoroacetic acid. The desired fractions were combined andfreeze-dried to provide the title compound. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 12.84 (s, 1H), 8.03 (dd, 4H), 7.79 (d, 1H), 7.62 (d,1H), 7.54 (dd, 1H), 7.51-7.41 (m, 2H), 7.36 (td, 2H), 7.33 (s, 1H), 6.98(dd, 1H), 4.96 (s, 2H), 4.42 (dd, 2H), 3.89 (t, 2H), 3.83 (s, 2H), 3.73(ddd, 2H), 3.57-3.38 (m, 2H), 3.31 (dt, 1H), 3.08 (dd, 1H), 3.02 (t,2H), 2.87 (tt, 1H), 2.81-2.54 (m, 2H), 2.10 (d, 3H), 1.51-0.91 (m, 12H),0.85 (s, 6H). MS (ESI) m/e 1005.2 (M+H)⁺.

1.38 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(H)-yl]-3-{1-[(3-{2-[{2-[(2-carboxyethyl)amino]ethyl}(2-sulfoethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (Compound W2.38) 1.38.16-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-(2-((2-((3-(tert-butoxy)-3-oxopropyl)amino)ethyl)(2-sulfoethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinicAcid

The title compound was prepared as described in Example 1.32.3,replacing Example 1.32.2 with Example 1.33.2.

1.38.26-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[{2-[(2-carboxyethyl)amino]ethyl}(2-sulfoethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid

The title compound was prepared as described in Example 1.6.2, replacingExample 1.6.1 with Example 1.38.1. ¹H NMR (501 MHz, dimethylsulfoxide-d₆) δ ppm 12.87 (s, 1H), 8.68 (s, 2H), 8.04 (d, 1H), 7.79 (d,1H), 7.62 (d, 1H), 7.53 (d, 1H), 7.42-7.50 (m, 2H), 7.33-7.40 (m, 2H),7.29 (s, 1H), 6.96 (d, 1H), 4.96 (s, 3H), 3.89 (t, 2H), 3.83 (s, 2H),3.66 (t, 2H), 3.31-3.53 (m, 8H), 3.18 (t, 2H), 3.02 (t, 2H), 2.95 (t,2H), 2.67 (t, 2H), 2.11 (s, 3H), 1.43 (s, 2H), 1.22-1.37 (m, 6H),0.98-1.19 (m, 6H), 0.87 (s, 6H). MS (APCI) m/e 971.0 (M+H)⁺.

1.39 Synthesis of3-{1-[(3,5-dimethyl-7-{2-[(3-phosphonopropyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-([1,3]thiazolo[4,5-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylicAcid (Compound W2.39) 1.39.1 Tert-butyl3-(1-((3-(2-((3-(di-tert-butoxyphosphoryl)propyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(thiazolo[4,5-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinate

Example 1.23.2 (520 mg) and Example 1.14.2 (175 mg) were dissolved indichloromethane (6 mL) and stirred at room temperature for two hours. Asuspension of sodium borohydride (32 mg) in methanol (1 mL) was added,and the mixture was stirred for 30 minutes. The reaction was added tosaturated aqueous NaHCO₃ solution and extracted with ethyl acetate. Theorganic layer was washed with brine and dried over sodium sulfate. Afterfiltration and concentration, purification by silica gel chromatography,eluting with a gradient of 0.5-5.0% methanol in dichloromethane, gavethe title compound. MS (ESI) m/e 1037.3 (M+H)⁺.

1.39.23-{1-[(3,5-dimethyl-7-(2-[(3-phosphonopropyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)-]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl)-6-[8-([1,3]thiazolo[4,5-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylicAcid

The title compound was prepared by substituting Example 1.39.1 forExample 1.2.8 in Example 1.2.9. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆)δ ppm 8.60 (dd, 1H), 8.52 (dd, 1H), 8.41 (br s, 2H), 7.65 (d, 1H) 7.48(d, 1H), 7.46 (d, 1H), 7.38 (m, 2H), 7.29 (s, 1H), 6.97 (d, 1H), 4.97(s. 2H), 3.89 (m, 2H), 3.83 (s, 2H), 3.56 (m, 2H), 3.02 (m, 6H), 2.11(s, 3H), 1.81 (m, 2H), 1.61 (m, 2H), 2.11 (s, 3H), 1.43 (s, 2H), 1.30(m, 4H), 1.14 (m, 4H), 1.04 (m, 2H), 0.87 (s, 6H). MS (ESI) m/e 869.2(M+H)⁺.

1.40 Synthesis of3-{1-[(3,5-dimethyl-7-{2-[(3-phosphonopropyl)amino]ethoxy}tricyclo[3.3.1.1j]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-([1,3]thiazolo[5,4-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylicAcid (Compound W2.40) 1.40.1 Tert-butyl3-(1-((3-(2-((3-(di-tert-butoxyphosphoryl)propyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(thiazolo[5,4-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinate

The title compound was prepared by substituting Example 1.22.2 forExample 1.23.2 in Example 1.39.1. MS (ESI) m/e 1037.3 (M+H)⁺.

1.40.23-{1-[(3,5-dimethyl-7-{2-[(3-phosphonopropyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-([1,3]thiazolo[5,4-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylicAcid

The title compound was prepared by substituting Example 1.40.1 forExample 1.2.8 in Example 1.2.9. ¹H NMR (500 MHz, dimethyl sulfoxide-d₆)δ ppm 8.52 (dd, 2H), 8.41 (br s, 2H), 8.17 (dd, 1H), 7.63 (m, 1H), 7.53(m, 2H), 7.46 (d, 1H), 7.38 (t, 1H), 7.30 (s, 1H), 6.98 (d, 1H), 4.96(s, 2H), 3.88 (m, 2H), 3.83 (s, 2H), 3.56 (t, 2H), 3.00 (m, 6H), 2.11(s, 3H), 1.81 (m, 2H), 1.60 (m, 2H), 1.43 (s, 2H), 1.31 (m, 4H), 1.14(m, 4H), 1.04 (m, 2H), 0.87 (s, 6H). MS (ESI) m/e 869.2 (M+H)⁺.

1.41 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-5-(carboxymethoxy)-3,4-dihydroisoquinolin-2(1H)-yl]-3-[1-({3,5-dimethyl-7-[2-(methylamino)ethoxy]tricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylicAcid (Compound W2.41) 1.41.1 methyl5-(2-(tert-butoxy)-2-oxoethoxy)-2-(6-(tert-butoxycarbonyl)-5-(1-((3-(2-((tert-butoxycarbonyl)(methyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)pyridin-2-yl)-1,2,3,4-tetrahydroisoquinoline-8-carboxylate

To a solution of Example 1.31.8 (163 mg) in N,N-dimethylformamide (10mL) was added tert-butyl 2-bromoacetate (58.6 mg), and K₂CO₃ (83 mg),and the reaction was stirred overnight. The mixture was diluted withethyl acetate (200 mL), washed with water and brine, and dried oversodium sulfate. Filtration and evaporation of the solvent gave a residuethat was purified by silica gel chromatography, eluting with 20% ethylacetate in heptane, to provide the title compound. MS (ESI) m/e 929.2(M+H)⁺.

1.41.25-(2-(tert-butoxy)-2-oxoethoxy)-2-(6-(tert-butoxycarbonyl)-5-(1-((3-(2-((tert-butoxycarbonyl)(methyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)pyridin-2-yl)-1,2,3,4-tetrahydroisoquinoline-8-carboxylicAcid

To a solution of Example 1.41.1 (3 g) in tetrahydrofuran (20 mL),methanol (10 mL) and water (10 mL) was added lithium hydroxidemonohydrate (30 mg). The mixture was stirred at room temperature for 24hours. The reaction mixture was neutralized with 2% aqueous HCl solutionand concentrated under vacuum. The residue was diluted with ethylacetate (800 mL), washed with water and brine, and dried over sodiumsulfate. Filtration and evaporation of the solvent provided the titlecompound. MS (ESI) me 914.5 (M+H)⁺.

1.41.36-[8-(1,3-benzothiazol-2-ylcarbamoyl)-5-(carboxymethoxy)-3,4-dihydroisoquinolin-2(1H)-yl]-3-[1-({3,5-dimethyl-7-[2-(methylamino)ethoxy]tricyclo[3.3.1.1^(3,7)-]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylicAcid

To a solution of Example 1.41.2 (183 mg) in N,N-dimethylformamide (4 mL)was added benzo[d]thiazol-2-amine (45.1 mg),fluoro-N,N,N′,N′-tetramethylformamidinium hexafluorophosphate (79 mg)and N,N-diisopropylethylamine (0.203 mL). The mixture was stirred at 60°C. overnight. The mixture was diluted with ethyl acetate (300 mL),washed with water and brine, and dried over sodium sulfate. Filtrationand evaporation of the solvent gave a residue that was dissolved indichloromethane/trifluoroacetic acid (1:1, 10 mL) and stirred overnight.The mixture was concentrated, and the residue was purified by reversephase HPLC using a Gilson system, eluting with 10-85% acetonitrile in inwater containing 0.1% v/v trifluoroacetic acid, to provide the titlecompound. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm 12.73 (s, 1H),8.30 (s, 2H), 7.99-8.07 (m, 1H), 7.75-7.79 (m, 1H), 7.70 (d, 1H),7.44-7.56 (m, 2H), 7.30-7.39 (m, 2H), 7.30 (s, 1H), 7.03 (t, 1H),6.87-6.93 (m, 1H), 4.98-5.18 (m 4H), 4.84 (s, 3H), 3.78-4.01 (m, 4H),3.55 (t, 2H), 2.77-3.07 (m, 4H), 2.53-2.61 (m, 3H), 2.04-2.16 (m, 3H),1.41 (s, 2H), 1.02-1.34 (m, 6H), 0.83-0.91 (m, 6H). MS (ESI) m/e 834.2(M+H)⁺.

1.42 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[(3-carboxypropyl)(piperidin-4-yl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (Compound W2.42) 1.42.1 Tert-butyl6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-(((1r,3r)-3-(2-((1-(tert-butoxycarbonyl)piperidin-4-yl)(4-methoxy-4-oxobutyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

A solution of Example 1.26.1 (0.169 g), methyl 4-oxobutanoate (0.024 g)and sodium triacetoxyborohydride (0.055 g) was stirred indichloromethane (2 mL) at room temperature. After 2 hours, the reactionwas diluted with dichloromethane (50 mL) and washed with saturatedaqueous sodium bicarbonate (10 mL). The organic layer was separated,dried over magnesium sulfate, filtered and concentrated. Silica gelchromatography, eluting with a gradient of 0.5-5%methanol/dichloromethane containing ammonia, provided the titlecompound. MS (ELSD) m/e 1085.5 (M+H)⁺.

1.42.26-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[(3-carboxypropyl)(piperidin-4-yl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid

A solution of Example 1.42.1 (0.161 g) in dichloromethane (0.5 mL) wastreated with trifluoroacetic acid (0.5 mL), and the reaction was stirredovernight. The reaction was concentrated, dissolved in methanol (0.6 mL)and treated with lithium hydroxide monohydrate (0.124 g) as a solutionin water (0.5 mL). After stirring for 1.5 hours, the reaction wasquenched with trifluoroacetic acid (0.229 mL) and diluted withN,N-dimethylformamide (0.5 mL). The mixture was purified by reversephase HPLC using a Gilson system, eluting with 10-60% acetonitrile inwater containing 0.1% v/v trifluoroacetic acid. The desired fractionswere combined and freeze-dried to provide the title compound. ¹H NMR(400 MHz, dimethyl sulfoxide-d₆) δ ppm 12.84 (s, 1H), 9.40 (s, 1H),8.89-8.79 (m, 1H), 8.57-8.41 (m, 1H), 8.03 (d, 1H), 7.80 (d, 1H), 7.62(d, 1H), 7.55-7.41 (m, 3H), 7.41-7.32 (m, 2H), 7.30 (s, 1H), 6.96 (d,1H), 4.96 (s, 2H), 3.89 (t, 2H), 3.83 (s, 2H), 3.44 (d, 2H), 3.26 (s,2H), 3.22-3.11 (m, 2H), 3.09-2.85 (m, 6H), 2.34 (t, 2H), 2.19 (d, 2H),2.10 (s, 3H), 1.95-1.71 (m, 5H), 1.44 (s, 2H), 1.39-1.27 (m, 4H),1.22-0.96 (m, 6H), 0.87 (s, 6H). MS (ESI) m/e 915.3 (M+H)⁺.

1.43 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (Compound W2.43) 1.43.1 Tert-butyl3-(1-((3-(2-hydroxyethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(methoxycarbonyl)naphthalen-2-yl)picolinate

To a solution of methyl7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-naphthoate (2.47 g) in1,4-dioxane (40 mL) and water (20 mL) was added Example 1.20.2 (4.2 g),bis(triphenylphosphine)palladium(II)dichloride (556 mg), and cesiumfluoride (3.61 g), and the reaction was stirred at reflux overnight. Themixture was diluted with ethyl acetate (400 mL) and washed with waterand brine, and dried over sodium sulfate. Filtration and evaporation ofthe solvent gave a residue that was purified by silica gelchromatography, eluting with 20% ethyl acetate in heptane followed by 5%methanol in dichloromethane, to provide the title compound. MS (ESI) m/e680.7 (M+H)⁺.

1.43.2 Tert-butyl3-(1-((3,5-dimethyl-7-(2-((methylsulfonyl)oxy)ethoxy)adamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(methoxycarbonyl)naphthalen-2-yl)picolinate

To a cooled (0° C.) solution of Example 1.43.1 (725 mg) indichloromethane (10 mL) and triethylamine (0.5 mL) was addedmethanesulfonyl chloride (0.249 mL), and the mixture was stirred for 4hours. The reaction mixture was diluted with ethyl acetate (200 mL) andwashed with water and brine, and dried over sodium sulfate. Filtrationand evaporation of the solvent gave the title product, which was used inthe next reaction without further purification. MS (ESI) m/e 759.9(M+H)⁺.

1.43.3 Tert-butyl3-(1-(((3-(2-azidoethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(methoxycarbonyl)naphthalen-2-yl)picolinate

To a solution of Example 1.43.2 (4.2 g) in N,N-dimethylformamide (30 mL)was added sodium azide (1.22 g), and the mixture was stirred for 96hours. The reaction mixture was diluted with ethyl acetate (600 mL),washed with water and brine, and dried over sodium sulfate. Filtrationand evaporation of the solvent provided the title compound. MS (ESI) m/e705.8 (M+H)⁺.

1.43.47-(5-(1-((3-(2-azidoethoxy)-5,7-dimethyladamantan-1-y)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(tert-butoxycarbonyl)pyridin-2-yl)-1-naphthoicAcid

To a solution of Example 1.43.3 (3.5 g) intetrahydrofuran/methanol/water (2:1:1, 30 mL) was added lithiumhydroxide monohydrate (1.2 g), and the mixture was stirred overnight.The reaction mixture was acidified with 1N aqueous HCl and was dilutedwith ethyl acetate (600 mL), washed with water and brine, and dried oversodium sulfate. Filtration and evaporation of the solvent provided thetitle compound. MS (ESI) m/e 691.8 (M+H)⁺.

1.43.5 Tert-butyl3-(1-((3-(2-azidoethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)naphthalen-2-yl)picolinate

To a solution of Example 1.43.4 (870 mg) in N,N-dimethylformamide (10mL) was added benzo[d]thiazol-2-amine (284 mg),fluoro-N,N,N′,N′-tetramethylformamidinium hexafluorophosphate (499 mg)and N,N-diisopropylethylamine (488 mg). The mixture was stirred at 60°C. for 3 hours. The reaction mixture was diluted with ethyl acetate (200mL) and washed with water and brine, and dried over sodium sulfate.Filtration and evaporation of the solvent provided the title compound.MS (ESI) m/e 824.1 (M+H)⁺.

1.43.6 Tert-butyl3-(1-((3-(2-aminoethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)naphthalen-2-yl)picolinate

To a solution of Example 1.43.5 (890 mg) in tetrahydrofuran (30 mL) wasadded Pd/C (90 mg). The mixture was stirred under 1 atmosphere ofhydrogen overnight. The reaction mixture was filtered, and the catalystwas washed with ethyl acetate. The solvent was evaporated to provide thetitle compound. MS (ESI) m/e 798.1 (M+H)⁺.

1.43.76-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid

To a solution of Example 1.43.6 (189 mg) in N,N-dimethylformamide (6 mL)was added 4-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylbutylethenesulfonate (106 mg). The mixture was stirred for 4 days. Themixture was diluted with ethyl acetate (300 mL) and washed with waterand brine and dried over sodium sulfate. After filtration andevaporation of the solvent, the residue was dissolved in trifluoroaceticacid (10 mL) and sat overnight. The trifluoroacetic acid was evaporatedunder vacuum, and the residue was dissolved in dimethylsulfoxide/methanol (1:1, 6 mL). The mixture was purified by reversephase HPLC (Gilson system), eluting with 10-85% acetonitrile in watercontaining 0.1% v/v trifluoroacetic acid, to give the title compound. ¹HNMR (400 MHz, dimethyl sulfoxid-d₆) δ ppm 13.09 (s, 1H), 9.02 (s, 1H),8.31-8.43 (m, 3H), 8.16-8.26 (m, 3H), 7.93-8.08 (m 3H), 7.82 (d, 1H),7.66-7.75 (m, 1H), 7.46-7.55 (m, 2H), 7.37 (t, 1H), 3.90 (s, 3H),3.17-3.28 (m, 2H), 3.07-3.16 (m, 2H), 2.82 (t, 2H), 2.24 (s, 3H), 1.44(s, 2H), 0.99-1.37 (m 12H), 0.87 (s, 6H). MS (ESI) m/e 849.1 (M+H)⁺.

1.44 Synthesis of3-{1-[(3-{2-[L-alpha-aspartyl(2-sulfoethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylicAcid (Compound W2.44) 1.44.16-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-(2-((S)-4-(tert-butoxy)-2-((tert-butoxycarbonyl)amino)-4-oxo-N-(2-sulfoethyl)butanamido)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinicAcid

To a cold (0° C.) solution of(S)-4-(tert-butoxy)-2-((tert-butoxycarbonyl)amino)-4-oxobutanoic acid(40.7 mg) and O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU. 40.1 mg,) in N,N-dimethylformamide (3 mL) wasadded N,N-diisopropylethylamine (98 μL). The reaction mixture wasstirred at room temperature for 1 hour, and Example 1.2.9 (60 mg) inN,N-dimethylformamide (1 mL) was added. The mixture was stirred for 1.5hours and was purified by reverse phase chromatography (C18 column),eluting with 20-90% acetonitrile in water containing 0.1% v/vtrifluoroacetic acid, to provide the title compound. MS (ESI) m/e 1123.4(M−H)⁻.

1.44.23-{1-[(3-{2-[L-alpha-aspartyl(2-sulfoethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylicAcid

Example 1.44.1 (100 mg) in dichloromethane (5 mL) was treated withtrifluoroacetic acid (1.5 mL) overnight. The reaction mixture wasconcentrated and purified by reverse phase chromatography (C18 column),eluting with 20-60% acetonitrile in water containing 0.1% v/vtrifluoroacetic acid, to provide the title compound. ¹H NMR (500 MHz,dimethyl sulfoxide-d₆) δ ppm 12.85 (s, 2H), 8.11-8.22 (m, 3H), 8.04 (d,1H), 7.79 (d, 1H), 7.62 (d, 1H), 7.41-7.54 (m, 3H), 7.32-7.39 (m, 2H),7.29 (s, 1H), 6.95 (d, 1H), 4.95 (s, 2H), 4.80 (s, 1H), 3.89 (t, 2H),3.81 (s, 2H), 3.55-3.71 (m, 2H), 3.01 (t, 4H), 2.74-2.86 (m, 1H),2.57-2.73 (m, 2H), 2.09 (s, 3H), 0.91-1.46 (m, 13H), 0.84 (s, 6H). MS(ESI) m/e %9.2 (M+H)⁺.

1.45 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[(1,3-dihydroxypropan-2-yl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (Compound W2.45) 1.45.1 Tert-butyl6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3,5-dimethyl-7-(2-(oxetan-3-ylamino)ethoxy)adamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

A solution of Example 1.2.7 (0.095 g), oxetan-3-one (10 mg) and sodiumtriacetoxyborohydride (0.038 g) was stirred in dichloromethane (1 mL) atroom temperature. After stirring overnight, the reaction mixture wasloaded directly onto silica gel and eluted using a gradient of 0.5-5%methanol in dichloromethane containing ammonia to give the titlecompound. MS (ELSD) me 858.4 (M+H)⁺.

1.45.26-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[(1,3-dihydroxypropan-2-yl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid

Example 1.45.1 was dissolved in dichloromethane (0.5 mL) and was treatedwith trifluoroacetic acid (0.5 mL) and stirred overnight. The reactionwas purified by reverse phase HPLC using a Gilson system, eluting with10-60% acetonitrile in water containing 0.1% v/v trifluoroacetic acid.The desired fractions were combined and freeze-dried to provide thetitle compound. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm 12.84 (s,1H), 8.19 (s, 2H), 8.02 (d, 1H), 7.78 (d, 1H), 7.61 (d, 1H), 7.53-7.40(m, 3H), 7.40-7.31 (m, 2H), 7.28 (s, 1H), 6.94 (d, 1H), 4.95 (s, 2H),3.87 (t, 2H), 3.82 (s, 2H), 3.67-3.62 (m, 4H), 3.22-3.14 (m, 1H),3.14-3.06 (m, 2H), 3.00 (t, 4H), 2.09 (s, 3H), 1.41 (s, 2H), 1.37-1.20(m, 4H), 1.20-0.95 (m, 6H), 0.85 (s, 6H). MS (ESI) nie 820.2 (M+H)⁺.

1.46 Synthesis of6-[5-(2-aminoethoxy)-8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[methyl(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (Compound W2.46) 1.46.16-[8-(1,3-benzothiazol-2-ylcarbamoyl)-5-(2-{[(benzyloxy)carbonyl]amino}ethoxy)-3,4-dihydroisoquinolin-2(1H)-yl]-3-[1-({3,5-dimethyl-7-[(2,2,7,7,13-pentamethyl-10,10-dioxido-3,3-diphenyl-4,9-dioxa-10λ⁶-thia-13-aza-3-silapentadecan-15-yl)oxy]tricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylicAcid

The title compound was prepared as described in Example 1.2.8, replacingExample 1.2.7 with Example 1.35.

1.46.26-[5-(2-aminoethoxy)-8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[methyl(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid

The title compound was prepared as described in Example 1.34.4,replacing Example 1.34.3 with Example 1.46.1. ¹H NMR (500 MHz, dimethylsulfoxide-d₆) δ ppm 12.74 (s, 2H), 8.% (s, 1H), 8.03 (d, 1H), 7.94 (s,3H), 7.72-7.81 (m, 2H), 7.53 (d, 1H), 7.47 (t, 1H), 7.35 (t, 1H), 7.28(s, 1H), 7.02 (t, 2H), 5.03 (s, 2H), 4.26 (t, 2H), 3.92 (t, 2H), 3.83(s, 2H), 3.23-3.38 (m, 4H), 3.13-3.25 (m, 1H), 2.82-3.00 (m, 4H), 2.78(d, 3H), 2.11 (s, 3H), 1.23-1.50 (m, 6H), 0.95-1.21 (m, 6H), 0.86 (s.6H). MS (ESI) m/e 927.2 (M+H)⁺.

1.47 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-5-{2-[(2-sulfoethyl)amino]ethoxy}-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[methyl(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (Compound W2.47) 1.47.16-[8-(1,3-benzothiazol-2-ylcarbamoyl)-5-[(2,2,7,7-tetramethyl-10,10-dioxido-3,3-diphenyl-4,9-dioxa-10λ⁶-thia-13-aza-3-silapentadecan-15-yl)oxy]-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[methyl(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid

The title compound was prepared as described in Example 1.2.8, replacingExample 1.2.7 with Example 1.46.2.

1.47.26-[8-(1,3-benzothiazol-2-ylcarbamoyl)-5-{2-[(2-sulfoethyl)amino]ethoxy}-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[methyl(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid

Example 1.47.1 (100 mg) in dichloromethane (5 mL) was treated withtrifluoroacetic acid (5 mL) overnight. The reaction mixture wasconcentrated and purified by reverse phase chromatography (C18 column),eluting with 20-60% acetonitrile in water containing 0.1% v/vtrifluoroacetic acid, to prvide the title compound. ¹H NMR (400 MHz,dimethyl sulfoxide-d₆) δ ppm m 12.74 (s, 1H), 8.96 (d, 1H), 8.64 (s,2H), 8.02 (d, 1H), 7.76 (dd, 2H), 7.41-7.57 (m, 2H), 7.24-7.40 (m, 2H),7.02 (t, 2H), 5.03 (s, 2H), 4.23-4.42 (m, 2H), 3.90 (t, 2H), 3.83 (s,2H), 3.25-3.40 (m, 6H). 3.12-3.24 (m, 2H), 2.81-3.01 (m, 6H), 2.78 (d,3H), 2.10 (s, 3H), 1.22-1.47 (m, 6H), 0.97-1.21 (m. 6H), 0.86 (s, 6H).MS (ESI) m/e 1035.3 (M+H)⁺.

1.48 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl){2-[(2-sulfoethyl)amino]ethyl}amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (Compound W2.48) 1.48.16-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{[2,2,7,7-tetramethyl-10,10-dioxido-3,3-diphenyl-16-(2-sulfoethyl)-4,9-dioxa-10λ⁶-thia-13,16-diaza-3-silaoctadecan-18-yl]oxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid

The title compound was prepared as described in Example 1.2.8, replacingExample 1.2.7 with Example 1.33.2.

1.48.26-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl){2-[(2-sulfoethyl)amino]ethyl}amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid

The title compound was prepared as described in Example 1.47.2,replacing Example 1.47.1 with Example 1.48.1. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 12.87 (s, 3H), 8.55 (s, 4H), 8.04 (d, 2H), 7.79 (d,2H), 7.62 (d, 1H), 7.40-7.56 (m 3H), 7.32-7.40 (m, 2H), 7.29 (s, 1H),6.96 (d, 2H), 4.96 (s, 3H), 3.89 (t, 2H), 3.83 (s, 2H), 3.47 (d, 2H),3.36 (s, 2H), 3.18-3.30 (m, 2H), 3.01 (t, 2H), 2.94 (t, 2H), 2.82 (t,2H), 2.11 (s, 3H), 1.26-1.49 (m, 6H), 0.96-1.20 (m, 6H), 0.87 (s. 6H).MS (ESI) m/e 1005.2 (M+H)⁺.

1.49 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-5-{2-[(2-carboxyethyl)amino]ethoxy}-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[methyl(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (Compound W2.49) 1.49.16-(8-(benzo[d]thiazol-2-ylcarbamoyl)-5-(2-((3-(tert-butoxy)-3-oxopropyl)amino)ethoxy)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3,5-dimethyl-7-(2-(methyl(2-sulfoethyl)amino)ethoxy)adamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinicAcid

The title compound was prepared as described in Example 1.32.3,replacing Example 1.32.2 with Example 1.46.2.

1.49.26-[8-(1,3-benzothiazol-2-ylcarbamoyl)-5-{2-[(2-carboxyethyl)amino]ethoxy}-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[methyl(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid

The title compound was prepared as described in Example 1.6.2, replacingExample 1.6.1 with Example 1.49.1. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 12.75 (s, 1H), 8.96 (s, 1H), 8.59 (s, 2H), 8.03 (d,1H), 7.72-7.82 (m, 2H), 7.54 (d, 1H), 7.43-7.51 (m, 2H), 7.35 (t, 1H),7.28 (s, 1H), 7.02 (dd, 2H), 5.02 (s, 2H), 4.34 (s, 2H), 3.93 (s, 2H),3.83 (s, 2H), 3.62 (s, 2H), 2.84-3.01 (m, 4f), 2.78 (d, 3H), 2.65-2.75(m, 2H), 2.11 (s, 3H), 1.20-1.45 (m, 7f), 0.95-1.21 (m, 6H), 0.86 (s,6H). MS (ESI) m/e 999.2 (M+H)⁺.

1.50 Synthesis of3-{1-[(3,5-dimethyl-7-{2-[(3-phosphonopropyl)(piperidin-4-yl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-([1,3]thiazolo[4,5-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylicAcid (Compound W2.50) 1.50.1 Tert-butyl3-(1-((3-(2-((1-(tert-butoxycarbonyl)piperidin-4-yl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(thiazolo[4,5-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinate

Example 1.23.2 (205 mg) was dissolved in dichloromethane (2.4 mL), andtert-butyl 4-oxopiperidine-1-carboxylate (51 mg) and sodiumtriacetoxyborohydride (75 mg) were added. The reaction was stirred atroom temperature for two hours. More dichloromethane was added, and thereaction was poured into to saturated aqueous NaHCO₃ solution. Theorganic layer was washed with brine and dried over sodium sulfate. Afterfiltration and concentration, the reside was purified by silica gelchromatography on a Grace Reveleris® Amino cartridge, eluting with agradient of 0.5 to 5.0% methanol in dichloromethane, to give the titlecompound. MS (ESI) m/e 986.3 (M+H)⁺.

1.50.23-{1-[(3,5-dimethyl-7-{2-[(3-phosphonopropyl)(piperidin-4-yl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-([1,3]thiazolo[4,5-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylicAcid

Example 1.50.1 (94 mg) was dissolved in dichloromethane (1 mL), thenExample 1.14.2 (25 mg) and sodium triacetoxyborohydride (30 mg) wereadded. The reaction was stirred at room temperature for four hours.Trifluoroacetic acid (1.5 mL) was added, and the reaction stirred atroom temperature overnight. The reaction mixture was concentrated andpurified by reverse phase chromatography (C18 column), eluting with10-90% acetonitrile in water containing 0.1% v/v trifluoroacetic acid,to provide the title compound as a trifluoroacetic acid salt. ¹H NMR(400 MHz, dimethyl sulfoxide-d₆) δ ppm 8.82 (br s, 1f) 8.60 (dd, 1H),8.52 (dd, 1H), 8.50 (br s. 1H), 7.66 (d, 1H), 7.50 (d, 1H), 7.46 (d,1H), 7.38 (m, 2H), 7.30 (s, 1H), 6.97 (d, 1H), 4.98 (s, 2H), 3.89 (t,2H), 3.83 (s, 2H) 3.69 (m, 2H), 3.61 (m, 1H), 3.44 (m, 2H) 3.23 (m, 4H),3.02 (t, 2H), 2.93 (m, 2H), 2.18 (m, 2H), 2.10 (s, 3H), 1.92 (m, 2H),1.83 (m, 2H), 1.64 (m, 2H), 1.44 (s, 2H), 1.31 (m, 4H), 1.14 (m, 4H),1.04 (m, 2H), 0.87 (s, 6H). MS (ESI) m/e 952.3 (M+H)⁺.

1.51 Synthesis of6-[4-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydro-2H-1,4-benzoxazin-6-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.‘’]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylic Acid(Compound W2.51) 1.51.1 Tert-butyl3-(1-((3-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-chloropicolinate

To a solution of Example 1.20.2 (3.2 g) in N,N-dimethylformamide (20 mL)was added imidazole (0.616 g) and chloro t-butyldimethylsilane (1.37 g).The mixture was stirred overnight. The reaction mixture was diluted withethyl acetate (300 mL), washed with water and brine, and dried oversodium sulfate. Filtration and evaporation of the solvent gave the crudeproduct that was purified by silica gel chromatography, eluting with 20%ethyl acetate in heptane, to provide the title compound. MS (ESI) m/e645.4 (M+H)⁺.

1.51.2 Tert-butyl3-(1-((3-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)picolinate

To a solution of6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazine(507 mg) in 1,4-dioxane (10 mL) and water (5 mL) was added Example1.51.1 (1.25 g), bis(triphenylphosphine)palladium(II)dichloride (136mg), and cesium fluoride (884 mg). The mixture was stirred at 120° C.under microwave conditions (Biotage. Initiator) for 20 minutes. Themixture was diluted with ethyl acetate (500 mL), washed with water andbrine, and dried over sodium sulfate. Filtration and evaporation of thesolvent gave a residue that was purified by silica gel chromatography,eluting with 20% ethyl acetate in heptane followed by 5% methanol indichloromethane, to provide the title compound. MS (ESI) m/e 744.1(M+H)⁺.

1.51.3 Tert-butyl6-(4-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-3-(1-((3-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-5,7-dimethyladamantan-1-y)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

To an ambient suspension of bis(2,5-dioxopyrrolidin-1-yl) carbonate (295mg) in acetonitrile (10 mL) was added benzo[d]thiazol-2-amine (173 mg),and the mixture was stirred for 1 hour. A solution of Example 1.51.2(710 mg) in acetonitrile (10 mL) was added, and the suspension wasvigorously stirred overnight. The mixture was diluted with ethyl acetate(300 mL), washed with water and brine, and dried over sodium sulfate.Filtration and evaporation of the solvent gave a residue that waspurified by silica gel chromatography, eluting with 20% ethyl acetate inheptane, to give the title compound. MS (ESI) m/e 920.2 (M+H)⁺.

1.51.4 Tert-butyl6-(4-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-3-(1-((3-(2-hydroxyethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

To a solution of Example 1.51.3 (1.4 g) in tetrahydrofuran (10 mL) wasadded tetrabutyl ammonium fluoride (1.0M in tetrahydrofuran, 6 mL). Themixture was stirred for 3 hours. The mixture was diluted with ethylacetate (300 mL), washed with water and brine, and dried over sodiumsulfate. Filtration and evaporation of the solvent gave title product,which was used in the next reaction without further purification. MS(ESI) m/e 806.0 (M+H)⁺.

1.51.5 Tert-butyl6-(4-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-3-(1-((3,5-dimethyl-7-(2-((methylsulfonyl)oxy)ethoxy)adamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

To a cooled (0° C.) solution of Example 1.51.4 (1.2 g) indichloromethane (20 mL) and triethylamine (2 mL) was addedmethanesulfonyl chloride (30 mg). The mixture was stirred for 4 hours.The reaction mixture was diluted with ethyl acetate (200 mL), washedwith water and brine, and dried over sodium sulfate. Filtration andevaporation of the solvent gave title product, which was used in thenext reaction without further purification. MS (ESI) m/e 884.1 (M+H)⁺.

1.51.6 Tert-butyl3-(1-((3-(2-azidoethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(4-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)picolinate

To a solution of Example 1.51.5 (1.5 g) in N,N-dimethylformamide (20 mL)was added sodium azide (331 mg). The mixture was stirred for 48 hours.The reaction mixture was diluted with ethyl acetate (200 mL), washedwith water and brine, and dried over sodium sulfate. Filtration andevaporation of the solvent gave a residue that was purified by silicagel chromatography, eluting with 20% ethyl acetate in dichloromethane,to provide the title compound. MS (ESI) m/e 831.1 (M+H)⁺.

1.51.7 Tert-butyl3-(1-((3-(2-aminoethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(4-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)picolinate

To a solution of Example 1.51.6 (1.5 g) in tetrahydrofuran (30 mL) wasadded Pd/C (10%. 200 mg). The mixture was stirred under 1 atmosphere ofhydrogen overnight. The reaction mixture was filtered, and the filtratewas concentrated under vacuum to give crude product. MS (ESI) m/e 805.1(M+H)⁺.

1.51.86-[4-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydro-2H-1,4-benzoxazin-6-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid

To a solution of Example 1.51.7 (164 mg) in N,N-dimethylformamide (10mL) and N,N-diisopropylethylamine (0.5 mL) was added4-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylbutyl ethenesulfonate (91mg). The mixture was stirred overnight. The reaction mixture was dilutedwith ethyl acetate (200 mL), washed with water and brine, and dried oversodium sulfate. Filtration and evaporation of the solvent gave a residuethat was dissolved in tetrahydrofuran (2 mL). Tetrabutyl ammoniumfluoride (1 mL. 1M in tetrahydrofuran) was added, and the mixture wasstirred overnight. The mixture was concentrated under vacuum, and theresidue was dissolved in dichloromethane/trifluoroacetic acid (1:1, 6mL), which was allowed to sit overnight. After evaporation of thesolvent, the residue was purified by reverse phase HPLC (Gilson system),eluting with 10-85% acetonitrile in water containing 0.1% v/vtrifluoroacetic acid, to provide the title compound. ¹H NMR (400 MHz,dimethyl sulfoxide-d₆) δ ppm 8.74 (s, 1H), 8.35 (s, 2H), 7.94-8.00 (m,1H), 7.86 (s, 1H), 7.71-7.82 (m, 2H), 7.46 (s, 1H), 7.34-7.44 (m, 2H),7.24 (t, 1H). 7.02 (d, 1H). 4.28-4.39 (m, 2H), 4.10-4.19 (m, 2H), 3.90(s, 3H), 3.55-3.61 (m, 4H), 3.21-3.30 (m, 3H), 3.07-3.16 (m, 3H), 2.23(s, 3H), 1.44 (s, 2H), 0.98-1.37 (m, 9H), 0.89 (s, 6H). MS (ESI) me856.1 (M+H)⁺.

1.52 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-5-(3-sulfopropoxy)-3,4-dihydroisoquinolin-2(1H)-yl]-3-[1-({3,5-dimethyl-7-[2-(methylamino)ethoxy]tricyclo[3.3.1.1^(3,7)-]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylicAcid (Compound W2.52) 1.52.1 methyl2-(6-(tert-butoxycarbonyl)-5-(1-((3-(2-((tert-butoxycarbonyl)(methyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)pyridin-2-yl)-5-(3-((2,2,2-trifluoro-1-(p-tolyl)ethoxy)sulfonyl)propoxy)-1,2,3,4-tetrahydroisoquinoline-8-carboxylate

To a solution of Example 1.31.8 (460 mg) in N,N-dimethylformamide (10mL) was added 2,2,2-trifluoro-1-(p-tolyl)ethyl 3-iodopropane-1-sulfonate(239 mg, prepared according to J. Org. Chem., 2013, 78, 711-716) andK₂CO3(234 mg), and the mixture was stirred overnight. The mixture wasdiluted with ethyl acetate (200 mL), washed with water and brine, anddried over sodium sulfate. Filtration and evaporation of the solventgave a residue that was purified by silica gel chromatography, elutingwith 20% ethyl acetate in heptane, to provide the title compound. MS(ESI) m/e 1018.5 (M+H)⁺.

1.52.22-(6-(tert-butoxycarbonyl)-5-(1-((3-(2-((tert-butoxycarbonyl)(methyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)pyridin-2-yl)-5-(3-((2,2,2-trifluoro-1-(p-tolyl)ethoxy)sulfonyl)propoxy)-1,2,3,4-tetrahydroisoquinoline-8-carboxylicAcid

To a solution of Example 1.52.1 (176 mg) in tetrahydrofuran (4 mL),methanol (3 mL) and water (3 mL) was added lithium hydroxide monohydrate(60 mg), and the mixture was stirred overnight. The mixture was thendiluted with ethyl acetate (200 mL), washed with 1N aqueous HC, waterand brine, and dried over sodium sulfate. Filtration and evaporation ofthe solvent gave the title product, which was used in the next reactionwithout further purification. MS (ESI) m/e 1095.2 (M+H)⁺.

1.52.3 Tert-butyl6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-5-(3-((2,2,2-trifluoro-1-(p-tolyl)ethoxy)sulfonyl)propoxy)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-(((2-((tert-butoxycarbonyl)(methyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

To a solution of Example 1.52.2 (117 mg) in dichloromethane (6 mL) wasadded benzo[d]thiazol-2-amine (19.27 mg).1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide hydrochloride (37 mg)and 4-(dimethylamino)pyridine (23.5 mg), and the mixture was stirredovernight. The reaction mixture was diluted with ethyl acetate (200 mL),washed with water and brine, and dried over sodium sulfate. Filtrationand evaporation of the solvent gave the title product. MS (ESI) m/e1226.1 (M+H)⁺.

1.52.46-[8-(1,3-benzothiazol-2-ylcarbamoyl)-5-(3-sulfopropoxy)-3,4-dihydroisoquinolin-2(1H)-yl]-3-[1-({3,5-dimethyl-7-[2-(methylamino)ethoxy]tricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylicAcid

Example 1.52.3 (130 mg) was dissolved in dichloromethane/trifluoroaceticacid (1:1, 6 mL) and stirred overnight. After evaporation of thesolvent, the residue was dissolved in N,N-dimethylformamide/water (1:1,12 mL) and purified by reverse phase HPLC (Gilson), eluting with 10 to85% acetonitrile in water containing 0.1% v/v trifluoroacetic acid, togive the title compound. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm12.68 (s, 1H), 8.13-8.32 (m, 2H), 8.01 (d, 1H), 7.75 (dd, 2H), 7.42-7.56(m, 2H), 7.29 (s, 1H), 7.28-7.34 (m, 1H), 7.00 (dd, 2H), 5.03 (s, 2H),4.19 (t, 2H), 3.83 (s, 3H), 3.50-3.57 (m, 4H), 2.95-3.05 (m, 2H), 2.81(t, 2H), 2.52-2.65 (m, 4H), 1.39 (s, 2H), 0.96-1.32 (m, 12H), 0.87 (s,6H). MS (ESI) m/e 898.3 (M+H)⁺.

1.53 Synthesis of3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[1-([1,3]thiazolo[4,5-b]pyridin-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]pyridine-2-carboxylicAcid (Compound W2.53) 1.53.1 Tert-butyl6-chloro-3-(1-((3-(2-hydroxyethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

The title compound was prepared as described in Example 1.51.4,replacing Example 1.51.3 with Example 1.51.1.

1.53.2 Tert-butyl6-chloro-3-(1-((3,5-dimethyl-7-(2-((methylsulfonyl)oxy)ethoxy)adamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

To a cooled (0° C.) solution of Example 1.53.1 (1.89 g) indichloromethane (30 mL) and triethylamine (3 mL) was addedmethanesulfonyl chloride (1.03 g), and the mixture was stirred for 4hours. The reaction mixture was diluted with ethyl acetate (200 mL),washed with water and brine, and dried over sodium sulfate. Filtrationand evaporation of the solvent gave the title product, which was used inthe next reaction without further purification.

1.53.4 Tert-butyl3-(1-((3-(2-aminoethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-chloropicolinate

Example 1.53.2 (2.2 g) was dissolved in 7N ammonia in methanol (40 mL),and the mixture was stirred at 80° C. under microwave conditions(Biotage Initiator) for 2 hours. The mixture was concentrated undervacuum and, and the residue was dissolved in ethyl acetate, washed withwater and brine, and dried over sodium sulfate. Filtration andevaporation of the solvent provided the title compound.

1.53.5 Tert-butyl6-chloro-3-[1-({3,5-dimethyl-7-[(2,2,7,7-tetramethyl-10,10-dioxido-3,3-diphenyl-4,9-dioxa-10λ⁶-thia-13-aza-3-silapentadecan-15-yl)oxy]tricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylate

To a solution of Example 1.53.3 (1.59 g) in N,N-dimethylformamide (30mL) was added 4-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylbutylethenesulfonate (1.6 g) and N,N-diisopropylethylamine (1 mL), and themixture was stirred for 4 days. The reaction mixture was dissolved inethyl acetate (400 mL), washed with water and brine, and dried oversodium sulfate. Filtration and evaporation of the solvent gave the titleproduct, which was used in the next reaction without furtherpurification. MS (ESI) m/e 976.8 (M+H)⁺.

1.53.6 Tert-butyl3-{1-[(3-{[13-(tert-butoxycarbonyl)-2,2,7,7-tetramethyl-10,10-dioxido-3,3-diphenyl-4,9-dioxa-10λ⁶-thia-13-aza-3-silapentadecan-15-yl]oxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-chloropyridine-2-carboxylate

To a solution of Example 1.53.4 (2.93 g) in tetrahydrofuran (50 mL) wasadded di-t-butyldicarbonate (0.786 g) and 4-(dimethylamino)pyridine (100mg), and the mixture was stirred overnight. The mixture was concentratedunder vacuum, and the residue was dissolved in ethyl acetate (300 mL),washed with 1N aqueous HCl solution, water and brine, and dried oversodium sulfate. Filtration and evaporation of the solvent gave a residuethat was purified by silica gel chromatography, eluting with 20% ethylacetate in heptane, to provide the title compound. MS (ESI) m/e 1076.9(M+H)⁺.

1.53.7 Tert-butyl3-{1-[(3-{[13-(tert-butoxycarbonyl)-2,2,7,7-tetramethyl-10,10-dioxido-3,3-diphenyl-4,9-dioxa-10λ⁶-thia-13-aza-3-silapentadecan-15-yl]oxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-(1,2,3,4-tetrahydroquinolin-7-yl)pyridine-2-carboxylate

To a solution of7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,2,3,4-tetrahydroquinoline(65 mg) in 1,4-dioxane (10 mL) and water (5 mL) was added Example 1.53.5(220 mg), bis(triphenylphosphine)palladium(II)dichloride (7 mg), andcesium fluoride (45.6 mg). The mixture was stirred at 120° C. for 30minutes under microwave conditions (Biotage Initiator). The mixture wasdiluted with ethyl acetate (200 mL), washed with water and brine, anddried over sodium sulfate. Filtration and evaporation of the solventgave a residue that was purified by silica gel chromatography, elutingwith 20% ethyl acetate in heptane, to give the title compound. MS (ESI)m/e 1173.9 (M+H)⁺.

1.53.83-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[1-([1,3]thiazolo[4,5-b]pyridin-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]pyridine-2-carboxylicAcid

To an ambient suspension of bis(2,5-dioxopyrrolidin-1-yl) carbonate(48.2 mg) in acetonitrile (10 mL) was addedthiazolo[4,5-b]pyridin-2-amine (34 mg), and the mixture was stirred for1 hour. A solution of Example 1.53.6 (220 mg) in acetonitrile (5 mL) wasadded, and the suspension was vigorously stirred overnight. The mixturewas diluted with ethyl acetate (200 mL), washed with water and brine,and dried over sodium sulfate. Filtration and evaporation of the solventgave a residue, which was dissolved in trifluoroacetic acid (10 mL) andstirred overnight. After evaporation of the solvent, the residue waspurified by reverse phase HPLC (Gilson system), eluting with 10-85%acetonitrile in water containing 0.1% v/v trifluoroacetic acid, toprovide the title compound. ¹H NMR (500 MHz, dimethyl sulfoxide-d₆) δppm 8.42-8.48 (m, 1H), 8.31-8.40 (m, 4H), 8.03 (d, 1H), 7.89 (d, 1H),7.80 (d, 1H), 7.47 (s, 1H), 7.26-7.37 (m, 2H), 3.93-4.02 (m, 3H), 3.90(s. 3H), 3.52-3.60 (m, 3H), 3.17-3.26 (m, 2H), 3.05-3.14 (m, 2H),2.76-2.89 (m, 5H), 2.23 (s, 3H), 1.90-2.01 (m, 2H), 1.44 (s, 2H),1.27-1.37 (m, 4H), 0.99-1.22 (m, 5H), 0.88 (s, 6H). MS (ESI) m/e 855.1(M+H)⁺.

1.54 Synthesis of3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-([1,3]thiazolo[4,5-b]pyridin-2-ylcarbamoyl)naphthalen-2-yl]pyridine-2-carboxylicAcid (Compound W2.54) 1.54.1 Tert-butyl3-{1-[(3-{[13-(tert-butoxycarbonyl)-2,2,7,7-tetramethyl-10,10-dioxido-3,3-diphenyl-4,9-dioxa-10λ⁶-thia-13-aza-3-silapentadecan-15-yl]oxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-(methoxycarbonyl)naphthalen-2-yl]pyridine-2-carboxylate

The title compound was prepared by substituting methyl7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-naphthoate for7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,2,3,4-tetrahydroquinolinein Example 1.53.6. MS (ESI) m/e 1226.6 (M+H)⁺.

1.54.27-[6-(tert-butoxycarbonyl)-5-{1-[(3-{[13-(tert-butoxycarbonyl)-2,2,7,7-tetramethyl-10,10-dioxido-3,3-diphenyl-4,9-dioxa-10′-thia-13-aza-3-silapentadecan-15-yl]oxy}-5,7-dimethyltricyclo[3.3.1.1⁰′7]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridin-2-yl]naphthalene-1-carboxylicAcid

To a solution of Example 1.54.1 (79 mg) in tetrahydrofuran (4 mL),methanol (3 mL) and water (3 mL) was added lithium hydroxide monohydrate(60 mg), and the mixture was stirred overnight. The reaction was dilutedwith ethyl acetate (200 mL), washed with 1N aqueous HCl, water andbrine, and dried over sodium sulfate. Filtration and evaporation of thesolvent gave the title product, which was used in the next step withoutfurther purification. MS (ESI) m/e 1211.6 (M+H)⁺.

1.54.33-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-([1,3]thiazolo[4,5-b]pyridin-2-ylcarbamoyl)naphthalen-2-yl]pyridine-2-carboxylicAcid

To a solution of Example 1.54.2 (60 mg) in dichloromethane (4 mL) wasadded thiazolo[4,5-b]pyridin-2-amine (7.56 mg),1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide hydrochloride (19 mg)and 4-(dimethylamino)pyridine (12.2 mg), and the mixture was stirredovernight. The reaction mixture was diluted with ethyl acetate (200 mL),washed with water and brine, and dried over sodium sulfate. Filtrationand evaporation of the solvent gave the title product, which wasdissolved in dichloromethane/trifluoroacetic acid (1:1, 6 mL) andstirred overnight. After evaporation of solvent, the residue wasdissolved in N,N-dimethylformamide/water (1:1, 12 mL) and purified byreverse phase HPLC (Gilson system), eluting with 10-85% acetonitrile inwater containing 0.1% trifluoroacetic acid, to give the title compound.¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm 13.42 (s, 1H), 9.05 (s,1H), 8.51-8.69 (m, 2H), 8.31-8.41 (m, 2H), 8.18-8.26 (m, 4H), 8.06 (d,1H), 7.97 (d, 1H), 7.68-7.79 (m, 1H), 7.49 (s, 1H), 7.40 (dd, 1H), 3.90(s, 3H), 3.18-3.29 (m, 3H), 3.07-3.15 (m, 2H), 2.82 (t, 3H), 2.24 (s,3H), 1.44 (s, 2H), 0.97-1.37 (m, 10H), 0.88 (s, 6H). MS (ESI) m/e 850.1(M+H)⁺.

1.55 Synthesis of(1ζ)-1-({2-[5-(1-{[3-(2-aminoethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)-6-carboxypyridin-2-yl]-8-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroisoquinolin-5-yl}methyl)-1,5-anhydro-D-glucitol(Compound W2.55) 1.55.1(2R,3R,4S,5R)-3,4,5-tris(methoxymethoxy)-2-((methoxymethoxy)methyl)-6-methylenetetrahydro-2H-pyran

The title compound was prepared according to J. R. Walker et al.,Bioorg. Med. Chem. 2006,14, 3038-3048. MS (ESI) m/e 370 (M+NH₄)⁺.

1.55.2 4-Bromo-3-cyanomethyl-benzoic Acid Methyl Ester

To a solution of trimethylsilanecarbonitrile (3.59 mL) intetrahydrofuran (6 mL) was added 1M tetrabutylammonium fluoride (26.8mL, 1 M in tetrahydrofuran) dropwise over 30 minutes. The solution wasstirred at room temperature for 30 minutes. Methyl4-bromo-3-(bromomethyl)benzoate (7.50 g) was dissolved in acetonitrile(30 mL) and was added to the first solution dropwise over 30 minutes.The solution was heated to 80° C. for 30 minutes and cooled. Thesolution was concentrated under reduced pressure, and the residue waspurified by silica gel chromatography, eluting with 20-30% ethyl acetatein heptanes, to provide the title compound.

1.55.3 3-(2-Aminoethyl)-4-bromobenzoic Acid Methyl Ester

Example 1.55.2 (5.69 g) was dissolved in tetrahydrofuran (135 mL), and 1M borane (in tetrahydrofuran. 24.6 mL) was added. The solution wasstirred at room temperature for 16 hours and was slowly quenched withmethanol and 1 M aqueous hydrochloric acid. 4 M Aqueous hydrochloricacid (150 mL) was added, and the solution was stirred at roomtemperature for 16 hours. The mixture was concentrated under reducedpressure, and the pH was adjusted to between 11 and 12 using solidpotassium carbonate. The solution was then extracted withdichloromethane (3×100 mL). The organic extracts were combined and driedover anhydrous sodium sulfate. The solution was filtered andconcentrated under reduced pressure, and the residue was purified bysilica gel chromatography, eluting with 10-20% methanol indichloromethane, to provide the title compound. MS (ESI) m/e 258.260(M+H)⁺.

1.55.4 4-Bromo-3-[2-(2,2,2-trifluoroacetylamino)-ethyl]-benzoic AcidMethyl Ester

Example 1.55.2 (3.21 g) was dissolved in dichloromethane (60 mL). Thesolution was cooled to 0° C., and triethylamine (2.1 mL) was added.Trifluoroacetic anhydride (2.6 mL) was added dropwise. The solution wasstirred at 0° C. for ten minutes, and the cooling bath was removed.After 1 hour, water (50 mL) was added, and the solution was diluted withethyl acetate (100 mL). 1 M Aqueous hydrochloric acid was added (50 mL),and the organic layer was separated, washed with 1 M aqueoushydrochloric acid, and washed with brine. The solution was dried withanhydrous sodium sulfate, filtered and concentrated under reducedpressure to provide the title compound. MS (ESI) me 371, 373 (M+H)⁺.

1.55.55-Bromo-2-(2,2,2-trifluoroacetyl)-1,2,3,4-tetrahydroisoquinoline-8-carboxylicAcid Methyl Ester

Example 1.55.4 (4.40 g) and paraformaldehyde (1.865 g) were placed in aflask and concentrated sulfuric acid (32 mL) was added. The solution wasstirred at room temperature for one hour. Cold water (120 mL) was added,and the solution was extracted with ethyl acetate (3×100 mL). Theextracts were combined, washed with saturated aqueous sodium bicarbonate(100 mL) and water (100 mL), and dried over anhydrous sodium sulfate.The mixture was filtered and concentrated under reduced pressure. Theresidue was purified by silica gel chromatography, eluting with 20-30%ethyl acetate in heptanes, to provide the title compound. MS (ESI) m/e366,368 (M+H)⁺.

1.55.6 Methyl2-(2,2,2-trifluoroacetyl)-5-(((3S,4R,5R,6R)-3,4,5-tris(methoxymethoxy)-6-((methoxymethoxy)methyl)tetrahydro-2H-pyran-2-yl)methyl)-1,2,3,4-tetrahydroisoquinoline-8-carboxylate

Example 1.55.1 (242 mg) was dissolved in tetrahydrofuran (7 mL) and9-borabicyclo[3.3.1]nonane (3.0 mL) was added dropwise. The solution wasrefluxed for 4.5 hours and allowed to cool to room temperature.Potassium phosphate (3M, 0.6 mL) was added, and the solution was stirredfor 10 minutes. The solution was then degassed and flushed with nitrogenthree times. Separately. Example 1.55.5 (239 mg) anddichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium (II)dichloromethane adduct (39 mg) were dissolved in N,N-dimethylformamide(7 mL), and the solution was degassed and flushed with nitrogen threetimes. The N,N-dimethylformamide solution was added dropwise to thetetrahydrofuran solution, and the mixture was stirred for 18 hours. HClsolution (0.1 M aqueous, 25 mL) was added, and the solution wasextracted with ethyl acetate (30 mL) three times. The organic extractswere combined, washed with brine, dried over anhydrous sodium sulfate,filtered and concentrated. The residue was purified by silica gelchromatography, eluting with 30-50% ethyl acetate in heptanes, to yieldthe title compound. MS (ESI) m/e 710 (M+NH₄)⁺.

1.55.7 Methyl5-(((3S,4R,5R,6R)-3,4,5-tris(methoxymethoxy)-6-((methoxymethoxy)methyl)tetrahydro-2H-pyran-2-yl)methyl)-1,2,3,4-tetrahydroisoquinoline-8-carboxylate

Example 1.55.6 (247 mg) was dissolved in methanol (1 mL),tetrahydrofuran (1 mL), and water (0.5 mL). Potassium carbonate (59 mg)was added, and the solution was stirred at room temperature for 16hours. The solution was diluted with ethyl acetate (10 mL) and washedwith saturated aqueous sodium bicarbonate (1 mL). The organic layer wasdried over anhydrous sodium sulfate, filtered and concentrated underreduced pressure to yield the title compound. MS (ESI) m/e 600 (M+H)⁺.

1.55.8 Methyl2-(5-bromo-6-(tert-butoxycarbonyl)pyridin-2-yl)-5-(((3S,4R,5R,6R)-3,4,5-tris(methoxymethoxy)-6-((methoxymethoxy)methyl)tetrahydro-2H-pyran-2-yl)methyl)-1,2,3,4-tetrahydroisoquinoline-8-carboxylate

The title compound was prepared by substituting Example 1.55.7 formethyl 1,2,3,4-tetrahydroisoquinoline-8-carboxylate in Example 1.1.11.MS (ESI) m/e 799, 801 (M-tert-butyl)⁺.

1.55.9 Methyl2-(6-(tert-butoxycarbonyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)-5-(((3S,4R,5R,6R)-3,4,5-tris(methoxymethoxy)-6-((methoxymethoxy)methyl)tetrahydro-2H-pyran-2-yl)methyl)-1,2,3,4-tetrahydroisoquinoline-8-carboxylate

The title compound was prepared by substituting Example 1.55.8 forExample 1.1.11 in Example 1.2.1. MS (ESI) m/e 903 (M+H)⁺. 933(M+MeOH—H)⁻.

1.55.102-((3-((4-iodo-5-methyl-1H-pyrazol-1-yl)methyl)-5,7-dimethyladamantan-1-yl)oxy)ethanamine

The title compound was prepared by substituting Example 1.13.1 forExample 1.10.4 in Example 1.10.5. MS (ESI) m/e 444 (M+H)⁺.

1.55.11 Tert-butyl(2-((3-((4-iodo-5-methyl-1H-pyrazol-1-yl)methyl)-5,7-dimethyladamantan-1-yl)oxy)ethyl)carbamate

The title compound was prepared by substituting Example 1.55.10 forExample 1.10.5 in Example 1.10.6. MS (ESI) m/e 544 (M+H)⁺. 488(M-tert-butyl)⁺, 542 (M−H)⁻.

1.55.12 Methyl2-(6-(tert-butoxycarbonyl)-5-(1-((3-(2-((tert-butoxycarbonyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)pyridin-2-yl)-5-(((3R,4S,5S,6S)-3,4,5-tris(methoxymethoxy)-6-((methoxymethoxy)methyl)tetrahydro-2H-pyran-2-yl)methyl)-1,2,3,4-tetrahydroisoquinoline-8-carboxylate

The title compound was prepared by substituting Example 1.55.9 forExample 1.2.1 and Example 1.55.11 for Example 1.13.3 in Example 1.13.4.MS (ESI) m/e 1192 (M+H)⁺.

1.55.132-(6-(tert-butoxycarbonyl)-5-(1-((3-(2-((tert-butoxycarbonyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)pyridin-2-yl)-5-(((3R,4S,5S,6S)-3,4,5-tris(methoxymethoxy)-6-((methoxymethoxy)methyl)tetrahydro-2H-pyran-2-yl)methyl)-1,2,3,4-tetrahydroisoquinoline-8-carboxylicAcid

The title compound was prepared by substituting Example 1.55.12 forExample 1.2.4 in Example 1.2.5. MS (ESI) m/e 1178 (M+H)⁺, 1176 (M−H)⁻.

1.55.14 Tert-butyl6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-5-(((3R,4S,5S,6S)-3,4,5-tris(methoxymethoxy)-6-((methoxymethoxy)methyl)tetrahydro-2H-pyran-2-yl)methyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-(2-((tert-butoxycarbonyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

The title compound was prepared by substituting Example 1.55.13 forExample 1.52.2 in Example 1.52.3. MS (ESI) m/e 1310 (M+H)⁺, 1308 (M−H)⁻.

1.55.15(1ζ)-1-({2-[5-(1-{[3-(2-aminoethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)-6-carboxypyridin-2-yl]-8-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroisoquinolin-5-yl}methyl)-1,5-anhydro-D-glucitol

The title compound was prepared by substituting Example 1.55.14 forExample 1.52.3 and 4M aqueous hydrochloric acid for trifluoroacetic acidin Example 1.52.4. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm 7.96(d, 1H), 7.73 (d, 1H), 7.58 (bs, 3H), 7.46 (d, 1H), 7.43-7.39 (m, 2H),7.30 (d, 1H), 7.27-7.25 (m, 2H), 6.88 (d, 1H), 4.90 (q, 2H), 3.76 (m,4H), 3.51 (m, 1H), 3.21 (d, 2H), 3.18 (d, 1H), 3.12 (m, 2H), 3.02 (m,4H), 2.93 (m, 4H), 2.83 (m, 2H), 2.59 (m, 2H), 2.03 (s, 3H), 1.44 (s,1H), 1.34 (s, 2H), 1.23 (q, 4H), 1.07 (m, 4H), 0.97 (q, 2H), 0.80 (s,6H). MS (ESI) m/e 922 (M+H)⁺. 920 (M−H)⁻.

1.56 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[(3-carboxypropyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (Compound W2.56) 1.56.1 Tert-butyl6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-(2-((4-(tert-butoxy)-4-oxobutyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

To a solution of Example 1.2.7 (0.103 g) and tert-butyl 4-bromobutanoate(0.032 g) in dichloromethane (0.5 mL) was addedN,N-diisopropylethylamine (0.034 mL) at 50° C. in a sealed amber vialovernight. The reaction was concentrated, dissolved in dimethylsulfoxide/methanol (1:1, 2 mL) and purified by reverse phase HPLC usinga Gilson system, eluting with 5-75% acetonitrile in water containing0.1% v/v trifluoroacetic acid. The desired fractions were combined andfreeze-dried to provide the title compound. MS (ESI) m/e 944.6 (M+1).

1.56.16-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[(3-carboxypropyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid

A solution of Example 1.56.1 (0.049 g) was dissolved in dichloromethane(1 mL) and treated with trifluoroacetic acid (0.5 mL) and the mixturewas stirred overnight. The reaction was concentrated, dissolved in a(1:1) N,N-dimethylformamide/water mixture (2 mL), and purified byreverse phase HPLC using a Gilson system, eluting with 5-75%acetonitrile in water containing 0.1% v/v trifluoroacetic acid. Thedesired fractions were combined and freeze-dried to provide the titlecompound. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm 1309-12.32 (m,2H), 8.31 (s, 2H), 8.03 (d, 1H), 7.79 (d, 1H), 7.62 (d, 1H), 7.54-7.40(m, 3H), 7.40-7.32 (m, 2H), 7.29 (s, 1H), 6.96 (d, 1H), 4.96 (s, 2H),3.89 (t, 2H), 3.83 (s, 2H), 3.55 (d, 2H), 3.02 (q, 4H), 2.92 (q, 2H),2.33 (t, 2H), 2.10 (s, 3H), 1.80 (p, 2H), 1.43 (s, 2H), 1.30 (q, 4H),1.21-0.95 (m, 6H), 0.87 (s, 6H). MS (ESI) m/e 832.3 (M+H)⁺.

1.57 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-3-(1-[(3,5-dimethyl-7-{2-[(3-phosphonopropyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylicAcid (Compound W2.57) 1.57.1 Tert-butyl3-(1-((3-(2-hydroxyethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(methoxycarbonyl)naphthalen-2-yl)picolinate

To a solution of methyl7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-naphthoate (2.47 g) in1,4-dioxane (40 mL) and water (20 mL) was added Example 1.20.2 (4.2 g),bis(triphenylphosphine)palladium(II)dichloride (556 mg), and cesiumfluoride (3.61 g). The mixture was refluxed overnight, diluted withethyl acetate (400 mL) and washed with water and brine. The organiclayer was dried over sodium sulfate, filtered, and concentrated. Theresidue was purified by silica gel chromatography, eluting with 20%ethyl acetate in dichloromethane and then with 5% methanol indichloromethane, to provide the title compound. MS (ESI) m/e 680.84(M+H)⁺.

1.57.2 Tert-butyl3-(1-((3,5-dimethyl-7-(2-((methylsulfonyl)oxy)ethoxy)adamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(methoxycarbonyl)naphthalen-2-yl)picolinate

To a cooled (0° C.) solution of Example 1.57.1 (725 mg) indichloromethane (10 mL) and triethylamine (0.5 mL) was addedmethanesulfonyl chloride (0.249 mL). The mixture was stirred at roomtemperature for 4 hours, diluted with ethyl acetate, and washed withwater and brine. The organic layer was dried over sodium sulfate,filtered, and concentrated to provide the title compound. MS (ESI) m/e758.93 (M+H)⁺.

1.57.3 Tert-butyl3-(1-((3-(2-azidoethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(methoxycarbonyl)naphthalen-2-yl)picolinate

To a solution of Example 1.57.2 (4.2 g) in N,N-dimethylformamide (30 mL)was added sodium azide (1.22 g). The mixture was stirred at roomtemperature for 96 hours, diluted with ethyl acetate (600 mL) and washedwith water and brine. The organic layer was dried over sodium sulfate,filtered, and concentrated to provide the title compound. MS (ESI) m/e704.86 (M+H)⁺.

1.57.47-(5-(1-((3-(2-azidoethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(tert-butoxycarbonyl)pyridin-2-yl)-1-naphthoicAcid

To a solution of Example 1.57.3 (3.5 g) in tetrahydrofuran/methanol/H₂O(2:1:1, 30 mL) was added lithium hydroxide monohydrate (1.2 g), and themixture was stirred at room temperature overnight. The reaction mixturewas acidified with 1N aqueous HC solution, diluted with ethyl acetate(6W mL) and washed with water and brine. The organic layer was driedover sodium sulfate, filtered, and concentrated to provide the titlecompound. MS (ESI) m/e 691.82 (M+H)⁺.

1.57.5 Tert-butyl3-(1-((3-(2-azidoethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)naphthalen-2-yl)picolinate

To a solution of Example 1.57.4 (870 mg) in N,N-dimethylformamide (10mL) was added benzo[d]thiazol-2-amine (284 mg),fluoro-N,N,N′N′-tetramethylformamidium hexafluorophosphate (499 mg) andN,N-diisopropylethylamine (488 mg). The mixture was stirred at 60° C.for 3 hours, diluted with ethyl acetate (200 mL), and washed with waterand brine. The organic layer was dried over sodium sulfate, filtered,and concentrated to provide the title compound. MS (ESI) m/e 824.02(M+H)⁺.

1.57.6 Tert-butyl3-(1-((3-(2-aminoethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)naphthalen-2-yl)picolinate

To a solution of Example 1.57.5 (890 mg) in tetrahydrofuran (30 mL) wasadded Pd/C (90 mg, 5%). The mixture was stirred under a hydrogenatmosphere at room temperature overnight, and filtered. The filtrate wasconcentrated to provide the title compound. MS (ESI) m/e 798.2 (M+H)⁺.

1.57.76-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-3-{1-[(3,5-dimethyl-7-{2-[(3-phosphonopropyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid

To a solution of Example 1.57.6 (137 mg) in dichloromethane (6 mL) wasadded Example 1.14.2 (43 mg). The mixture was stirred at roomtemperature for 1.5 hours, and a solution of NaBH₄ (26 mg) in methanol(2 mL) was added. The mixture was stirred at room temperature for 2hours, diluted with ethyl acetate (200 mL) and washed with 2N aqueousNaOH solution, water and brine. The organic layer was dried over sodiumsulfate, filtered, and concentrated. The residue was dissolved indichloromethane (5 mL) and treated with trifluoroacetic acid (5 mL)overnight. The reaction mixture was concentrated. The residue waspurified by reverse phase HPLC (Gilson system), eluting with a gradientof 10-85% acetonitrile in water containing 0.1% v/v trifluoroacetic acidsolution, to provide the title compound. ¹H NMR (500 MHz, dimethylsulfoxide-d₆) δ 9.03 (s, 1H), 8.48-8.35 (m, 3H), 8.29-8.16 (m, 3H), 8.08(dd, 1H), 8.03 (dd. 1H), 7.94 (d. 1H), 7.82 (d, 1H), 7.71 (dd, 1H),7.53-7.47 (m, 2H), 7.38 (td, 1H), 4.81-0.53 (m, 89H). MS (ESI) m/e 863.2(M+H)⁺.

1.58 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3-(2-{[4-(beta-D-glucopyranosyloxy)benzyl]amino}ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl)-5-methyl}-1H-pyrazol-4-yl)pyridine-2-carboxylicAcid (Compound W2.58)

To a solution of Example 1.3.1 (44.5 mg) in tetrahydrofuran (2 mL) andacetic acid (0.2 mL) was added4-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)benzaldehyde(17 mg) and MgSO₄ (300 mg). The mixture was stirred at room temperaturefor 1 hour before the addition of sodium cyanoborohydride on resin (300mg). The mixture was stirred at room temperature overnight and filtered.The filtrate was concentrated, and the residue was purified by reversephase HPLC (Gilson system), eluting with a gradient of 10-85%acetonitrile in water containing 0.1% v/v trifluoroacetic acid solution,to provide the title compound. MS (ESI) m/e 1015.20 (M+H)⁺.

1.59 Synthesis of3-(1-{[3-(2-{[4-(beta-D-allopyranosyloxy)benzyl]amino}ethoxy)-5,7-dimethyltricyclo[3.3.1.1¹⁷]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylicAcid (Compound W2.59)

To a solution of Example 1.3.1 (44.5 mg) in tetrahydrofuran (2 mL) andacetic acid (0.2 mL) was added4-(((2S,3R,4S5,S6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)benzaldehyde(17 mg) and MgSO₄ (300 mg), and the mixture was stirred at roomtemperature for 1 hour before the addition of sodium cyanoborohydride onresin (300 mg). The mixture was stirred at room temperature overnightand filtered. The filtrate was concentrated, and the residue waspurified by reverse phase HPLC (Gilson system), eluting with a gradientof 10-85% acetonitrile in water containing 0.1% v/v trifluoroaceticacid, to provide the title compound. MS (ESI) m/e 1015.20 (M+H)⁺.

1.60 Synthesis of3-{1-[(3-{2-[azetidin-3-yl(2-sulfoethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylicAcid (Compound W2.60) 1.60.1 Tert-butyl6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-(2-((1-(tert-butoxycarbonyl)azetidin-3-yl)(2-((4-(tert-butyldiphenylsilyl)hydroxy-2,2-dimethylbutoxy)sulfonyl)ethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

A solution of Example 1.2.8 (0.075 g), tert-butyl3-oxoazetidine-1-carboxylate (0.021 g) and sodium triacetoxyborohydride(0.025 g) in dichloromethane (0.5 mL) was stirred at room temperatureovernight. The reaction was loaded onto silica gel and eluted with 0-10%methanol in dichloromethane to give the title compound. MS (ESI) m/e1403.9 (M+1).

1.60.23-{1-[(3-{2-[azetidin-3-yl(2-sulfoethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylicAcid

A solution of Example 1.60.1 (0.029 g) in dichloromethane (1 mL) wastreated with trifluoroacetic acid (1 mL) and stirred overnight. Thereaction was concentrated, dissolved in 1:1dimethyl sulfoxide/methanol(2 mL), and the mixture was purified by reverse phase HPLC using aGilson system, eluting with 10-80% acetonitrile in water containing 0.1%v/v trifluoroacetic acid. The desired fractions were combined andfreeze-dried to provide the title compound. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 12.86 (s, 1H), 8.81 (s, 2H), 8.04 (d, 1H), 7.79 (d,1H), 7.62 (d, 1H), 7.52 (d, 1H), 7.50-7.46 (m, 1H), 7.44 (d, 1H),7.40-7.33 (m, 2H), 7.30 (s, 1H), 6.96 (d, 1H), 4.96 (s, 2H), 4.37 (q,1H), 4.27 (s, 2H), 4.11 (s, 2H), 3.89 (t, 2H), 3.83 (s, 2H), 3.58-3.54(m, 2H), 3.32 (t, 2H), 3.24 (s, 2H), 3.01 (t, 2H), 2.85 (t, 2H), 2.10(s, 3H), 1.48-0.97 (m, 12H), 0.87 (s, 6H). MS (ESI) m/e 909.2 (M+H)⁺.

1.61 Synthesis of3-{1-[(3-(2-[(3-aminopropyl)(2-sulfoethyl)amino]ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylicAcid (Compound W2.61) 1.61.16-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-(2-((3-((tert-butoxycarbonyl)amino)propyl)(2-sulfoethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinicAcid

The title compound was prepared using the procedure for Example 1.33.1,replacing tert-butyl (2-oxoethyl)carbamate with tert-butyl(3-oxopropyl)carbamate. MS (ESI) m/e 1011.5 (M+H).

1.61.23-{1-[(3-{2-[(3-aminopropyl)(2-sulfoethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridine-2-carboxylicAcid

The title compound was prepared as described in Example 1.6.2, replacingExample 1.6.1 with Example 1.61.1. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 12.87 (s, 1H), 9.10 (s, 1H), 8.04 (d, 1H), 7.88-7.67(m, 4H), 7.62 (d, 1H), 7.57-7.40 (m, 3H), 7.36 (td, 2H), 6.96 (d, 1H),4.96 (s, 2H), 4.05-3.78 (m, 4H), 3.41-3.08 (m, 3H), 2.94 (tt, 6H), 2.11(s, 3H), 1.92 (t, 2H), 1.53-0.95 (m, 11H), 0.87 (s, 6H). MS (ESI) m/e911.3 (M+H)⁺.

1.62 Synthesis of6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]-3-{1-[(3-{2-[(2-carboxyethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (Compound W2.62) 1.62.1 Tert-butyl3-(1-((3-(2-((3-(tert-butoxy)-3-oxopropyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-chloropicolinate

To an ambient solution of Example 1.53.3 (521 mg) in ethanol (10 mL) wasadded triethylamine (3 mL) followed by tert-butyl acrylate (2 mL). Themixture was stirred at room temperature for 3 hours and thenconcentrated to dryness. The residue was dissolved in ethyl acetate (200mL), and the solution was washed with water and brine. The organic layerwas dried over sodium sulfate, filtered and concentrated under reducedpressure to give the title compound, which was used in the next reactionwithout further purification. MS (ESI) m/e 657.21 (M+H)⁺.

1.62.2 Tert-butyl3-(1-((3-(2-((3-(tert-butoxy)-3-oxopropyl)(tert-butoxycarbonyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-chloropicolinate

To a solution of Example 1.62.1 (780 mg) in tetrahydrofuran (10 mL) wasadded di-tert-butyl dicarbonate (259 mg) followed by a catalytic amountof 4-dimethylaminopyridine. The reaction was stirred at room temperaturefor 3 hours and then concentrated to dryness. The residue was dissolvedin ethyl acetate (200 mL), and the solution was washed with saturatedaqueous NaHCO solution, water and brine. The organic layer was driedover sodium sulfate, filtered and concentrated under reduced pressure.The residue was purified by chromatography on silica gel, eluting with20% ethyl acetate in heptane, to give the title compound. MS (ESI) m/e757.13 (M+H)⁺.

1.62.3 Tert-butyl3-(1-((3-(2-((3-(tert-butoxy)-3-oxopropyl)(tert-butoxycarbonyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(1,2,3,4-tetrahydroquinolin-7-yl)picolinate

To a solution of7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,2,3,4-tetrahydroquinoline(234 mg) in 1,4-dioxane (10 mL) and water (5 mL) was added Example1.62.2 (685 mg), bis(triphenylphosphine)palladium(II)dichloride (63.2ng), and cesium fluoride (410 mg). The mixture was heated to 120° C. for30 minutes by microwave irradiation (Biotage Initiator). The reactionwas quenched by the addition of ethyl acetate and water. The layers wereseparated, and the organic layer was washed with brine, dried oversodium sulfate, filtered and concentrated under reduced pressure. Theresidue was purified by chromatography on silica gel, eluting with 20%ethyl acetate in heptane, to give the title compound. MS (ESI) m/e854.82 (M+H)⁺.

1.62.4 Tert-butyl6-(1-(benzo[d]thiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl)-3-(1-((3-(2-((3-(tert-butoxy)-3-oxopropyl)(tert-butoxycarbonyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

To an ambient suspension of bis(2,5-dioxopyrrolidin-1-yl) carbonate (150mg) in acetonitrile (10 mL) was added benzo[d]thiazol-2-amine (88 mg),and the mixture was stirred for 1 hour. A solution of Example 1.62.3(500 mg) in acetonitrile (2 mL) was added, and the suspension wasvigorously stirred overnight. The reaction was quenched by the additionof ethyl acetate and water. The layers were separated, and the organiclayer was washed with brine, dried over sodium sulfate, filtered andconcentrated under reduced pressure. The residue was purified bychromatography on silica gel, eluting with 20% ethyl acetate indichloromethane, to give the title compound. MS (ESI) m/e 1030.5 (M+H)⁺.

1.62.56-[1-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]-3-{1-[(3-{2-[(2-carboxyethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid

To an ambient solution of Example 1.62.4 (110 mg) in dichloromethane(0.53 mL) was added trifluoroacetic acid (0.53 mL). The reaction wasstirred overnight and was concentrated to a viscous oil. The residue wasdissolved in dimethyl sulfoxide/methanol (1:1, 2 mL) and purified byreverse phase HPLC (Gilson system), eluting with 10-55% acetonitrile in0.1% trifluoroacetic acid in water, to give the title compound. ¹H NMR(400 MHz, dimethyl sulfoxide-d₆) δ ppm 13.10 (s, 3H), 8.37 (s, 1H), 8.26(s, 2H), 7.98 (d, 1H), 7.86-7.71 (m, 3H), 7.44 (s, 1H), 7.39-7.31 (m,1H), 7.26 (d. 1H), 7.19 (t, 1H), 3.92 (d, 2H), 3.87 (s, 2H), 3.55 (t,2H), 3.17-3.00 (m, 4H), 2.80 (t, 2H), 2.62 (t, 2H), 2.19 (s, 3H),1.95-1.88 (m, 2H), 1.43 (s, 2H), 1.33-1.25 (m, 4H), 1.18-1.11 (m, 4H),1.09-0.97 (m, 2H), 0.85 (s, 6H). MS (ESI) m/e 818.0 (M+H)⁺.

1.63 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[(N6,N6-dimethyl-L-lysyl)(methyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (W2.63)

A solution of(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-(dimethylamino)hexanoicacid (0.029 g) and1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate (0.028 g) was stirred together inN,N-dimethylformamide (0.5 mL) with N,N-diisopropylamine (0.035 mL).After stirring for 5 minutes, the solution was added to Example 1.13.7(0.051 g) and stirring was continued at room temperature overnight. Tothe reaction was added diethylamine (0.070 mL), and the reaction wasstirred for 2 hours. The reaction was diluted with N,N-dimethylformamide(1 mL), water (0.5 ml), and 2,2,2-trifluoroacetic acid (0.103 ml) thenpurified via reverse-phase HPLC using a gradient of 10% to 90%acetonitrile/water. The product containing fractions were collected andlyophilized to give the title compound. ¹H NMR (500 MHz, dimethylsulfoxide-d₆) δ 9.59 (s, 1H), 8.41 (s, 1H), 8.12 (t, 3H), 8.01 (d, 1H),7.85 (dd, 1H), 7.81 (d, 1H), 7.77 (dd, 1H), 7.47 (s, 1H), 7.38 (t, 1H),7.30 (d, 1H), 7.22 (t, 1H), 3.97 (t, 2H), 3.89 (s, 2H), 3.49 (dt, 4H),3.06 (s, 2H), 2.99 (q, 2H), 2.88 (s, 2H), 2.84 (t, 2H), 2.75 (d, 6H),2.22 (s, 3H), 2.00-1.90 (m, 2H), 1.84-1.52 (m, 4H), 1.48-0.95 (m, 14H),0.87 (d, 6H). MS (ESI) m/e 916.2 (M+H)⁺.

1.64 Synthesis of3-{1-[(3-{2-[(3-aminopropyl)(methyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]pyridine-2-carboxylicAcid (W2.64) 1.64.16-(1-(benzo[d]thiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl)-3-(1-((3-(2-((3-((tert-butoxycarbonyl)amino)propyl)(methyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinicAcid

A solution of Example 1.21.5 (100 mg), N,N-diisopropylethylamine (68.9μL) and tert-butyl (3-oxopropyl)carbamate (68.4 mg) in dichloromethane(3 mL) was stirred at ambient temperature for 2 hours, and NaCNBH₄ (8.27mg) was added. The reaction was stirred at ambient temperatureovernight. Methanol (1 mL) and water (0.2 mL) were added. The resultingmixture was stirred for 10 minutes and concentrated. The residue wasdissolved in dimethyl sulfoxide and purified by reverse-phase HPLC on aGilson system (C18 column), eluting with 30-80% acetonitrile in 0.1%trifluoroacetic acid water solution, to provide the title compound as atrifluoroacetic acid salt. MS (ESI) m/e 459.4 (M+2H)²⁺.

1.64.23-{1-[(3-{2-[(3-aminopropyl)(methyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]pyridine-2-carboxylicAcid

Example 1.64.1 (100 mg) in dichloromethane (4 mL) at 0° C. was treatedwith trifluoroacetic acid (1 mL) for 1 hour, and the mixture wasconcentrated. The residue was purified by reverse phase HPLC (C18column), eluting with a gradient of 10-60% acetonitrile in 0.1%trifluoroacetic acid water solution, to provide the title compound as atrifluoroacetic acid salt. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ 69.38 (s, 1H), 8.37 (s, 1H), 7.98 (d, 1H), 7.90-7.69 (m, 6H), 7.44 (s,2H), 7.35 (td. 1H), 7.27 (d, 1H), 7.22-7.16 (m, 1H), 3.94 (d, 2H), 3.87(s, 2H), 3.64 (t, 2H), 3.28-2.98 (m, 4H), 2.87-2.70 (m, 8H), 2.19 (s,3H), 1.90 (dp, 4H), 1.43 (s, 2H), 1.36-1.22 (m, 4H), 1.15 (s, 4H),1.08-0.95 (m, 2H), 0.86 (s, 6H). MS (ESI) m/e 817.6 (M+H)⁺.

1.65 Synthesis of3-{1-[(3-{2-[azetidin-3-yl(methyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]pyridine-2-carboxylicacid (W2.65) 1.65.16-(1-(benzo[d]thiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl)-3-(1-((3-(2-((1-(tert-butoxycarbonyl)azetidin-3-yl)(methyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinicAcid

The title compound was prepared using the procedure described in Example1.64.1, substituting tert-butyl (3-oxopropyl)carbamate with tert-butyl3-oxoazetidine-1-carboxylate. MS (ESI) m/e 915.3 (M+H)⁺.

1.65.23-{1-[(3-{2-[azetidin-3-yl(methyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]pyridine-2-carboxylicAcid

The title compound was prepared using the procedure in Example 1.64.2,substituting Example 1.64.1 with Example 1.65.1. ¹H NMR (400 MHz,dimethyl sulfoxide-d₆) δ 9.01 (s, 2H), 8.37 (s, 1H), 7.98 (d, 1H),7.86-7.70 (m, 3H), 7.44 (s, 2H), 7.34 (td. 1H), 7.27 (d, 1H), 7.23-7.15(m, 1H), 4.22 (s, 4H), 4.07 (s, 2H), 3.93 (t, 2H), 3.58 (t, 2H), 3.11(s, 2H), 2.80 (t, 2H), 2.68 (s, 3H), 2.19 (s, 3H), 1.92 (p, 2H), 1.42(s, 2H), 1.30 (s, 4H), 1.15 (s, 4H), 1.09-0.% (m 2H), 0.85 (s, 6H). MS(ESI) m/e 815.5 (M+H)⁺.

1.66 Synthesis ofN6-(37-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-yl)-L-lysyl-N-[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]-L-alaninamide(W2.66) 1.66.1(S)-6-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-((tert-butoxycarbonyl)amino)hexanoicAcid

To a solution of (S)-6-amino-2-((tert-butoxycarbonyl)amino)hexanoic acid(8.5 g) in a mixture of 5% aqueous NaHCO₃ solution (300 mL) and dioxane(40 mL), chilled in an ice bath, was added dropwise a solution of(9H-fluoren-9-yl)methyl pyrrolidin-1-yl carbonate (11.7 g) in dioxane(40 mL). The reaction mixture was allowed to warm to room temperatureand was stirred for 24 hours. Three additional vials were set up asdescribed above. After the reaction was completed, all four reactionmixtures were combined, and the organic solvent was removed undervacuum. The aqueous residue was acidified to pH 3 with aqueoushydrochloric acid solution (1N) and then extracted with ethyl acetate(3×500 mL). The combined organic layers were washed with brine, driedover magnesium sulfate, filtered, and concentrated under vacuum to givea crude compound which was recrystallized from methyl tert-butyl etherto afford the title compound. ¹H NMR (400 MHz, chloroform-d) δ 11.05(br. s., 1H), 7.76 (d, 2H), 7.59 (d, 2H), 7.45-7.27 (m, 4H), 6.52-6.17(m, 1H), 5.16-4.87 (m, 1H), 4.54-4.17 (m, 4H), 3.26-2.98 (m, 2H),1.76-1.64 (m, 1H), 1.62-1.31 (m, 14H).

1.66.2 Tert-butyl 17-hydroxy-3,6,9,12,15-pentaoxaheptadecan-1-oate

To a solution of 3,6,9,12-tetraoxatetradecane-1,14-diol (40 g) intoluene (800 mL) was added portion-wise potassium tert-butoxide (20.7g). The mixture was stirred at room temperature for 30 minutes.Tert-butyl 2-bromoacetate (36 g) was added dropwise to the mixture. Thereaction was stirred at room temperature for 16 hours. Two additionalvials were set up as described above. After the reactions werecompleted, all three reaction mixtures were combined. Water (500 mL) wasadded to the combined mixture, and the mixture was concentrated to 1 L.The mixture was extracted with dichloromethane and was washed withaqueous 1N potassium tert-butoxide solution (1 L). The organic layer wasdried over Na₂SO₄, filtered and concentrated to obtain crude product,which was purified by silica gel column chromatography, eluting withdichloromethane:methanol 50:1, to obtain the title compound. ¹H NMR (400MHz, chloroform-d) δ 4.01 (s, 2H), 3.75-3.58 (m, 21H), 1.46 (s, 9H).

1.66.3 Tert-butyl 17-(tosyloxy)-3,6,9,12,15-pentaoxaheptadecan-1-oate

To a solution of Example 1.66.2 (30 g) in dichloromethane (500 mL) wasadded dropwise a solution of 4-methylbenzene-1-sulfonyl chloride (19.5g) and triethylamine (10.3 g) in dichloromethane (500 mL) at 0° C. undera nitrogen atmosphere. The mixture was stirred at room temperature for18 hours and was poured into water (100 mL). The solution was extractedwith dichloromethane (3×150 mL), and the organic layer was washed withhydrochloric acid (6N. 15 mL) then NaHCO₃ (5% aqueous solution, 15 mL)followed by water (20 mL). The organic layer was dried over Na₂SO₄,filtered and concentrated to obtain a residue, which was purified bysilica gel column chromatography, eluting with petroleum ether:ethylacetate 10:1 to dichloromethane:methanol 5:1, to obtain the titlecompound. ¹H NMR (400 MHz, chloroform-d) δ 7.79 (d, 2H), 7.34 (d, 2H),4.18-4.13 (m, 2H), 4.01 (s, 2H), 3.72-3.56 (m, 18H), 2.44 (s, 3H), 1.47(s, 9H).

1.66.4 2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-oicAcid

To a solution of 2,5,8,11,14,17-hexaoxanonadecan-19-ol (32.8 g) intetrahydrofuran (300 mL) was added sodium hydride (1.6 g) at 0° C. Themixture was stirred at room temperature for 4 hours. A solution ofExample 1.66.3 (16 g) in tetrahydrofuran (300 mL) was added dropwise atroom temperature to the reaction mixture. The resulting reaction mixturewas stirred at room temperature for 16 hours and then water (20 mL) wasadded. The mixture was stirred at room temperature for another 3 hoursto complete the tert-butyl ester hydrolysis. The final reaction mixturewas concentrated under vacuum to remove the organic solvent. The aqueousresidue was extracted with dichloromethane (2×150 mL). The aqueous layerwas acidified to pH 3 and then extracted with ethyl acetate (2×150 mL).The aqueous layer was concentrated to obtain crude product, which waspurified by silica gel column chromatography, eluting with a gradient ofpetroleum ether:ethyl acetate 1:1 to dichloromethane:methanol 5:1, toobtain the title compound. ¹H NMR (400 MHz, chloroform-d) δ 4.19 (s,2H), 3.80-3.75 (m, 2H), 3.73-3.62 (m, 40H), 3.57 (dd, 2H), 3.40 (s, 3H).

1.66.5(43S,46S)-43-((tert-butoxycarbonyl)amino)-46-methyl-37,44-dioxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-38,45-diazaheptatetracontan-47-oicAcid

Example 1.66.5 was synthesized using standard Fmoc solid phase peptidesynthesis procedures and a 2-chlorotrytil resin. 2-Chlorotrytil resin(12 g, 100 mmol),(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoic acid (10 g,32.1 mmol) and N,N-diisopropylethylamine (44.9 mL, 257 mmol) inanhydrous, sieve-dried dichloromethane (100 mL) was shaken at 14° C. for24 hours. The mixture was filtered and the cake was washed withdichloromethane (3×500 mL), dimethylformamide (2×250 mL) and methanol(2×250 mL) (for 5 minutes for each step). To the above resin was added20% piperidine/dimethylformamide (100 mL) to remove the Fmoc group. Themixture was bubbled with nitrogen for 15 minutes and then filtered. Theresin was washed with 20% piperidine/dimethylformamide (100 mL) anotherfive times (5 minutes each step), and washed with dimethylformamide(5×100 mL) to give the deprotected, L-Ala loaded resin.

To a solution of Example 1.66.1 (9.0 g) in N,N-dimethylformamide (50 mL)was added hydroxybenzotriazole (3.5 g),2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminiumhexafluorophosphate (9.3 g) and N,N-diisopropylethylamine (8.4 mL). Themixture was stirred at 20° C. for 30 minutes. The above mixture wasadded to the D-Ala loaded resin and mixed by bubbling with nitrogen atroom temperature for 90 minutes. The mixture was filtered and the resinwas washed with dimethylformamide (5 minutes each step). To the aboveresin was added approximately 20% piperidine/N,N-dimethylformamide (100mL) to remove the Fmoc group. The mixture was bubbled with nitrogen for15 minutes and filtered. The resin was washed with 20%piperidine/dimethylformamide (100 mL) for another five times (5 minutesfor each step), and finally washed with dimethylformamide (5×100 mL).

To a solution of Example 1.66.4 (11.0 g) in N,N-dimethylformamide (50mL) was added hydroxybenzotriazole (3.5 g).2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminiumhexafluorophosphate (9.3 g) and N,N-diisopropylethylamine (8.4 mL), andthe mixture was added to the resin and mixed by bubbling with nitrogenat room temperature for 3 hours. The mixture was filtered and theresidue was washed with dimethylformamide (5×100 mL), dichloromethane(8×100 mL) (5 minutes for each step).

To the final resin was added 1% trifluoroacetic acid/dichloromethane(100 mL) and nitrogen was bubbled through for 5 minutes. The mixture wasfiltrated and the filtrate was collected. The cleavage operation wasrepeated for four times. The combined filtrate was brought to pH 7 byNaHCO3 and washed with water. The organic layer was dried over Na₂SO4,filtered and concentrated to obtain the title compound. ¹H NMR (400 MHz,methanol-d₄) δ 4.44-4.33 (m, 1H), 4.08-4.00 (m, 1H), 3.98 (s, 2H),3.77-3.57 (m, 42H), 3.57-3.51 (m, 2H), 3.36 (s, 3H), 3.25 (t, 2H), 1.77(br. s., 1H), 1.70-1.51 (m, 4H), 1.44 (s, 9H), 1.42-1.39 (m, 3H).

1.66.6 Tert-butyl6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-(((43S,46S)-43-((tert-butoxycarbonyl)amino)-46-methyl-37,44,47-trioxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-38,45,48-triazapentacontan-50-yl)oxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

Example 1.66.5 (123 mg, 0.141 mmol), was mixed with1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate (58.9 mg) and N,N-diisopropylethylamine(0.049 mL) in N-methyl-2-pyrrolidone (1 mL) for 10 minutes and thenadded to a solution of Example 1.2.7 (142 mg) andN,N-diisopropylethylamine (0.049 mL) in N-methyl-2-pyrrolidone (1.5 mL).The reaction mixture was stirred at room temperature for two hours. Thecrude reaction mixture was purified by reverse phase HPLC using a Gilsonsystem and a C18 25×100 mm column, eluting with 5-85% acetonitrile inwater containing 0.1% v/v trifluoroacetic acid. The product fractionswere lyophilized to give the title compound. MS (LC/MS) m/e 1695.5(M+H)⁺.

1.66.73-(1-((3-(((43S,46S)-43-amino-46-methyl-37,44,47-trioxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-38,45,48-triazapentacontan-50-yl)oxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid

Example 1.66.6 (82 mg) was treated with 1 mL of trifluoroacetic acid atroom temperature for 30 minutes. The solvent was evaporated under agentle stream of nitrogen, and the residue was purified by reverse phaseHPLC using a Gilson system and a C18 25×100 mm column, eluting with5-85% acetonitrile in water containing 0.1% v/v trifluoroacetic acid.The product fractions were lyophilized to give the title compound as thetrifluoroacetic acid salt. ¹H NMR (400 MHz, dimethyl sulfoxide-d) δ ppm12.86 (s, 1H), 8.04 (dd, 4H), 7.64 (dt, 2H), 7.55-7.41 (m, 3H), 7.36 (q,2H), 6.95 (d, 1H), 4.96 (s, 2H), 4.40-4.27 (m, 1H), 3.93-3.72 (m, 7H),3.59-3.47 (m 42H), 3.33-3.27 (m, 3H), 3.23 (s, 5H), 3.05 (dt, 5H), 2.10(s, 3H), 1.72-1.64 (m, 2H), 1.48-1.36 (m, 4H), 1.35-1.16 (m, 10H),1.16-0.94 (m, 6H), 0.84 (d, 6H). MS (ESI) m/e 751.8 (2M+H)².

1.67 Synthesis of methyl6-[4-(3-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]amino}propyl)-1H-1,2,3-triazol-1-yl]-6-deoxy-beta-L-glucopyranoside(W2.67) 1.67.16-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3,5-dimethyl-7-(2-(pent-4-yn-1-ylamino)ethoxy)adamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinicAcid

To a solution of tert-butyl3-(1-((3-(2-aminoethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinate(85 mg) in tetrahydrofuran (2 mL) was added pent-4-ynal (8.7 mg), aceticacid (20 mg, 0.318) and anhydrous sodium sulfate (300 mg). The mixturewas stirred at room temperature for 1 hour. Sodium triacetoxyborohydride(45 mg) was added to the reaction mixture. The mixture was stirred atroom temperature overnight. The reaction mixture was diluted with ethylacetate (200 mL), washed with water and brine, and dried over anhydroussodium sulfate. Filtration and evaporation of the solvent gave crudeproduct, which was dissolved in dichloromethane (5 mL) andtrifluoroacetic acid (3 mL). The mixture was stirred at room temperatureovernight. After evaporation of the solvent, the residue was dissolvedin dimethyl sulfoxide/methanol (1:1, 3 mL) and purified by reverse-phaseHPLC on a Gilson system (C18 column), eluting with 20-80% acetonitrilein water containing 0.1% trifluoroacetic acid, to give the titlecompound. MS (APCI) m/e 812.2 (M+H)⁺.

1.67.2 methyl6-[4-(3-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1J]dec-1-yl}oxy)ethyl]amino}propyl)-1H-1,2,3-triazol-1-yl]-6-deoxy-beta-L-glucopyranoside

To a solution of(2R,3R,4S,5S,6S)-2-azido-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (8.63 mg) in t-BuOH (2 mL) and water (1 mL) was added Example1.67.1 (20 mg), copper (II) sulfate pentahydrate (2.0 mg) and sodiumascorbate (5 mg). The mixture was heated for 20 minutes at 100° C. undermicrowave conditions (Biotage Initiator). LiOH H₂O (50 mg) was added tothe mixture, which was stirred at room temperature overnight. Themixture was neutralized with trifluoroacetic acid and purified byreverse-phase HPLC on a Gilson system (C18 column), eluting with 20-80%acetonitrile in water containing 0.1% trifluoroacetic acid, to give thetitle compound. MS (APCI) m/e 1032.2 (M+H)⁺.

1.68 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-3-{1-[(3-{2-[(2-carboxyethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid 1.68.12-((3,5-dimethyl-7-((5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)methyl)adamantan-1-y)oxy)ethanol(W2.68)

To a solution of2-((3-((4-iodo-5-methyl-1H-pyrazol-1-yl)methyl)-5,7-dimethyladamantan-1-yl)oxy)ethanol(8.9 g) and PdCl₂(dppf)-CH₂CH₂ adduct(([1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (1:1), 818mg) in acetonitrile (120 mL) was added trimethylamine (10 mL) and4,4,5,5-tetramethyl-1,3,2-dioxaborolane (12.8 mL). The mixture wasstirred at reflux overnight. The mixture was cooled to room temperatureand used in the next reaction without further work up. MS (ESI) m/e467.3 (M+Na)⁺.

1.68.2 Tert-butyl6-chloro-3-(1-((3-(2-hydroxyethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

To a solution of tert-butyl 3-bromo-6-chloropicolinate (6.52 g) intetrahydrofuran (100 mL) and water (20 mL) was added Example 1.68.1(9.90 g),(1S,3R,5R,7S)-1,3,5,7-tetramethyl-8-tetradecyl-2,4,6-trioxa-8-phosphaadamantane(0.732 g), tris(dibenzylideneactone)dipalladium(0) (Pd₂(dba)₃, 1.02 g),and K₃PO₄ (23.64 g). The mixture was stirred at reflux overnight. Themixture was concentrated under reduced pressure, the residue wasdissolved in ethyl acetate (500 mL), washed with water and brine, anddried over anhydrous sodium sulfate. Filtration and evaporation of thesolvent gave crude product, which was purified by silica gelchromatography eluting with 20 to 40% ethyl acetate in dichloromethaneto give the title compound. MS (ESI) m/e 530.3 (M+H)⁺.

1.68.3 Tert-butyl6-chloro-3-(1-((3,5-dimethyl-7-(2-((methylsulfonyl)oxy)ethoxy)adamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

To a cooled (0° C.) solution of Example 1.68.2 (3.88 g) indichloromethane (30 mL) and triethylamine (6 mL) was addedmethanesulfonyl chloride (2.52 g). The mixture was stirred at roomtemperature for 4 hours. The reaction mixture was diluted with ethylacetate (400 mL), washed with water and brine, and dried over anhydroussodium sulfate. Filtration and evaporation of the solvent gave the crudeproduct (4.6 g), which was used in the next reaction without furtherpurification. MS (ESI) m/e 608.1 (M+H)⁺.

1.68.4 Tert-butyl3-{1-[(3-{2-[bis(tert-butoxycarbonyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-chloropyridine-2-carboxylate

To a solution of Example 1.68.3 (151 mg) in N,N-dimethylformamide (3 mL)was added di-tert-butyl iminodicarboxylate (54 mg). The mixture wasstirred at room temperature overnight. The reaction mixture was dilutedwith ethyl acetate (200 mL), washed with water and brine, and dried overanhydrous sodium sulfate. Filtration and evaporation of the solvent gavethe title compound, which was used in the next step without furtherpurification. MS (ESI) m/e 729.4 (M+H)⁺.

1.68.57-(6-(tert-butoxycarbonyl)-5-(1-((3-(2-((tert-butoxycarbonyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)pyridin-2-yl)-1-naphthoicAcid

To a solution of methyl7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-naphthoate (257 mg) in1,4-dioxane (10 mL) and water (5 mL) was added Example 1.68.4 (60 mg),bis(triphenylphosphine)palladium(II) dichloride (57.8 mg), and CsF (375mg). The mixture was stirred at 120° C. for 30 minutes under microwaveconditions (Biotage Initiator). The mixture was diluted with ethylacetate (200 mL), washed with water and brine, and dried over anhydroussodium sulfate. Filtration and evaporation of the solvent gave crudeproduct, which was purified by silica gel chromatography, eluting with20% ethyl acetate in heptane to give a di-ester intermediate. Theresidue was dissolved in tetrahydrofuran (10 mL), methanol (5 mL) andwater (5 mL) and LiOH H₂O (500 mg) was added, and the mixture wasstirred at room temperature overnight. The mixture was acidified with 2Naqueous HCl, dissolved in 400 mL of ethyl acetate, washed with water andbrine, and dried over anhydrous sodium sulfate. Filtration andevaporation of the solvent gave the title compound. MS (APCI) m/e 765.3(M+H)⁺.

1.68.63-(1-((3-(2-aminoethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)naphthalen-2-yl)picolinicAcid

To a solution of Example 1.68.5 (500 mg) in dichloromethane (10 mL) wasadded benzo[d]thiazol-2-amine (98 mg),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (251 mg) and4-dimethylaminopyridine (160 mg). The mixture was stirred at roomtemperature overnight. The reaction mixture was diluted with ethylacetate (400 mL), washed with water and brine, and dried over anhydroussodium sulfate. Filtration and evaporation of the solvent gave a residuethat was dissolved in dichloromethane and trifluoroacetic acid (10 mL,1:1). After stirring overnight, the solution was concentrated underreduced pressure. The residue was dissolved in N,N-dimethylformamide (12mL) and purified by reverse-phase HPLC (using a Gilson system and a C18column, eluting with 20-80% acetonitrile in water containing 0.1%trifluoroacetic acid) to give the title compound. MS (ESI) m/e 741.2(M+H)⁺.

1.68.76-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-3-{1-[(3-{2-[(2-carboxyethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid

To a solution of Example 1.68.6 (35 mg) in N,N-dimethylformamide (4 mL)was added tert-butyl acrylate (120 mg) and H₂O (138 mg). The mixture wasstirred at room temperature overnight. The reaction mixture was dilutedwith ethyl acetate (400 mL), washed with water and brine, and dried overanhydrous sodium sulfate. Filtration and evaporation of the solvent gavea residue that was dissolved in dichloromethane and trifluoroacetic acid(10 mL, 1:1). After 16 hours, the mixture was concentrated under reducedpressure. The residue was dissolved in N,N-dimethylformamide (2 mL) andpurified by reverse-phase HPLC on a Gilson system (C18 column), elutingwith 20-80% acetonitrile in water containing 0.1% trifluoroacetic acid,to give the title compound. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δppm 13.08 (s, 1H), 8.99 (d, 1H), 8.43-8.24 (m, 4H), 8.24-8.11 (m, 3H),8.04 (d, 1H), 7.99 (d, 1H), 7.90 (d, 1H), 7.78 (d, 1H), 7.74-7.62 (m,1H), 7.53-7.43 (m 2H), 7.35 (q, 1H), 3.87 (s, 2H), 3.08 (dp, 4H), 2.62(t, 2H), 2.20 (s, 3H), 1.43 (s, 2H), 1.29 (q, 4H), 1.14 (s, 4H), 1.03(q, 2H), 0.85 (s, 6H).

1.69 Synthesis of6-[5-(1,3-benzothiazol-2-ylcarbamoyl)quinolin-3-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid 1.69.1 methyl 3-bromoquinoline-5-carboxylate (W2.69)

To a solution of 3-bromoquinoline-5-carboxylic acid (2 g) in methanol(30 mL) was added concentrated H₂SO₄ (5 mL). The solution was stirred atreflux overnight. The mixture was concentrated under reduced pressure.The residue was dissolved in ethyl acetate (300 mL) and washed withaqueous Na₂CO₃ solution, water and brine. After drying over anhydroussodium sulfate, filtration and evaporation of the solvent gave the titlecompound. MS (ESI) m/e 266 (M+H)⁺.

1.69.2 Methyl3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)quinoline-5-carboxylate

To a solution of Example 1.69.1 (356 mg) in N,N-dimethylformamide (5 mL)was added PdCl₂(dppf)-CH₂CH₂ adduct([1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (1:1), 55mg) potassium acetate (197 mg) and bis(pinacolato)diboron (510 mg). Themixture was stirred at 60° C. overnight. The mixture was cooled to roomtemperature and used in the next reaction without further work up. MS(ESI) m/e 339.2 (M+Na)⁺.

1.69.3 Methyl3-[5-{1-[(3-{2-[bis(tert-butoxycarbonyl}amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl)-6-(tert-butoxycarbonyl)pyridin-2-yl]quinoline-5-carboxylate

To a solution of Example 1.69.2 (626 mg) in 1,4-dioxane (10 mL) andwater (5 mL) was added Example 1.68.4 (1.46 g),bis(triphenylphosphine)palladium(II) dichloride (140 mg), and CsF (911mg). The mixture was stirred at 120° C. for 30 minutes under microwaveconditions (Biotage Initiator). The mixture was diluted with ethylacetate (200 mL), washed with water and brine, dried over anhydroussodium sulfate, filtered and concentrated. The residue was purified bysilica gel chromatography, eluting with 20% ethyl acetate in heptane (1L) to give the title compound. MS (ESI) m/e 880.3 (M+H)⁺.

1.69.43-(6-(tert-butoxycarbonyl)-5-(1-((3-(2-((tert-butoxycarbonyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)pyridin-2-yl)quinoline-5-carboxylicAcid

To a solution of Example 1.69.3 (1.34 g) in tetrahydrofuran (10 mL),methanol (5 mL) and water (5 mL) was added LiOH H₂O (120 mg), and themixture was stirred at room temperature overnight. The mixture wasacidified with 2N aqueous HCl, diluted with ethyl acetate (400 mL),washed with water and brine, and dried over anhydrous sodium sulfate.Filtration and evaporation of the solvent gave the title compound. MS(APCI) m/e 766.3 (M+H)⁺.

1.69.53-(1-((3-(2-aminoethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(5-(benzo[d]thiazol-2-ylcarbamoyl)quinolin-3-yl)picolinicAcid

To a solution of Example 1.69.4 (200 mg) in dichloromethane (10 mL) wasadded benzo[d]thiazol-2-amine (39.2 mg),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (50 mg) and4-dimethylaminopyridine (32 mg). The mixture was stirred at roomtemperature overnight. The reaction mixture was diluted with ethylacetate (200 mL), washed with water and brine, dried over anhydroussodium sulfate, filtered, and concentrated. The residue was dissolved indichloromethane and trifluoroacetic acid (10 mL, 1:1), and the reactionwas stirred overnight. The mixture was concentrated, and the residue wasdissolved in N,N-dimethylformamide (12 mL) and purified by reverse-phaseHPLC on a Gilson system (C18 column), eluting with 20-80% acetonitrilein water containing 0.1% trifluoroacetic acid, to give the titlecompound. MS (ESI) m/e 742.1 (M+H)⁺.

1.69.66-[5-(1,3-benzothiazol-2-ylcarbamoyl)quinolin-3-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid

To a solution of Example 1.69.5 (36 mg) in N,N-dimethylformamide (2 mL)was added 4-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylbutylethenesulfonate (22 mg) and H₂O (0.3 mL)). The mixture was stirred atroom temperature for 3 hours. The reaction mixture was diluted withdichloromethane and trifluoroacetic acid (10 mL, 1:1) and stirredovernight. The mixture was concentrated, and the residue was dissolvedin N,N-dimethylformamide (4 mL) and purified by reverse-phase HPLC on aGilson system (C18 column), eluting with 20-80/acetonitrile in watercontaining 0.1% trifluoroacetic acid, to give the title compound. ¹H NMR(400 MHz, dimethyl sulfoxide-d₆) δ ppm 13.19 (s, 2H), 9.70 (d, 1H), 9.40(s, 1H), 8.31 (d, 2H), 8.16 (d, 1H), 8.06 (d, 1H), 8.01 (d, 1H),7.98-7.88 (m, 1H), 7.80 (d, 1H), 7.52-7.43 (m, 2H), 7.37 (q, 1H), 3.89(s, 2H), 3.22 (p, 2H), 3.10 (q, 2H), 2.80 (t, 2H), 2.23 (s, 3H), 1.43(s, 2H), 1.30 (q, 4H), 1.23-1.10 (m, 4H), 1.04 (q, 2H), 0.87 (s, 6H). MS(ESI) m/e 850.2 (M+H)⁺.

1.70 Synthesis of6-[4-(1,3-benzothiazol-2-ylcarbamoyl)quinolin-6-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (W2.70) 1.70.1 ethyl6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)quinoline-4-carboxylate

To a solution of ethyl 6-bromoquinoline-4-carboxylate (140 mg) inN,N-dimethylformamide (2 mL) was added PdCl₂(dppf)-CH₂C2 adduct(([1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (1:1),20.42 mg), potassium acetate (147 mg) and bis(pinacolato)diboron (190mg). The mixture was stirred at 60° C. overnight. The mixture was cooledto room temperature and used in the next reaction without further workup. MS (ESI) m/e 328.1 (M+H)⁺.

1.70.2 Ethyl6-[5-{1-[(3-{2-[bis(tert-butoxycarbonyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-(tert-butoxycarbonyl)pyridin-2-yl]quinoline-4-carboxylate

To a solution of Example 1.70.1 (164 mg) in 1,4-dioxane (10 mL) andwater (5 mL) was added Example 1.68.4 (365 mg),bis(triphenylphosphine)palladium(II) dichloride (35 mg), and CsF (228mg). The mixture was stirred at 120° C. for 30 minutes under microwaveconditions (Biotage Initiator). The mixture was diluted with ethylacetate (200 mL), washed with water and brine, dried over anhydroussodium sulfate, filtered and concentrated. The residue was purified bysilica gel chromatography, eluting with 20% ethyl acetate in heptane (1L) to give the title compound. MS (ESI) m/e 894.3 (M+H)⁺.

1.70.36-(6-(tert-butoxycarbonyl)-5-(1-((3-(2-((tert-butoxycarbonyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)pyridin-2-yl)quinoline-4-carboxylicAcid

To a solution of Example 1.70.2 (3.1 g) in tetrahydrofuran (20 mL),methanol (10 mL) and water (10 mL) was added LiOH H₂O (240 mg). Themixture was stirred at room temperature overnight. The mixture wasacidified with 2N aqueous HCl and diluted with ethyl acetate (400 mL).The organic layer was washed with water and brine and dried overanhydrous sodium sulfate. Filtration and evaporation of the solvent gavethe title compound. MS (ESI) m/e 766.3 (M+H)⁺.

1.70.43-(1-((3-(2-aminoethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(4-(benzo[d]thiazol-2-ylcarbamoyl)quinolin-6-yl)picolinicAcid

To a solution of Example 1.70.3 (4.2 g) in dichloromethane (30 mL) wasadded benzo[d]thiazol-2-amine (728 mg).1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (1.40 g) and4-dimethylaminopyridine (890 mg), and the mixture was stirred at roomtemperature overnight. The reaction mixture was diluted with ethylacetate (500 mL), washed with water and brine, and dried over anhydroussodium sulfate. Filtration and evaporation of the solvent gave a residuethat was dissolved in dichloromethane and trifluoroacteic acid (10 mL,1:1) and stirred overnight. The mixture was concentrated, and theresidue was dissolved in N,N-dimethylformamide (4 mL) and purified byreverse-phase HPLC on a Gilson system (C18 column), eluting with 20-80%acetonitrile in water containing 0.1% trifluoroacetic acid, to give thetitle compound. MS (ESI) m/e 742.2 (M+H)⁺.

1.70.56-[4-(1,3-benzothiazol-2-ylcarbamoyl)quinolin-6-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid

To a solution of Example 1.70.4 (111 mg) in N,N-dimethylfornamide (4 mL)was added4-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylbutylethenesulfonate (67mg). N,N-diisopropylethylamine (0.2 mL) and H₂O (0.3 mL). The mixturewas stirred at room temperature for 3 hours. The reaction mixture wasdiluted with dichloromethane and trifluoroacetic acid (10 mL, 1:1) andstirred overnight. The mixture was concentrated, and the residue wasdissolved in N,N-dimethylformamide (4 mL) and purified by reverse-phaseHPLC on a Gilson system (C18 column), eluting with 20-80/o acetonitrilein water containing 0.1% trifluoroacetic acid, to give the titlecompound. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm 13.31 (s, 1H),9.10 (d, 1H), 8.91 (s, 1H), 8.58 (dd, 1H), 8.47-8.16 (m, 4H), 8.06 (dd,1H), 7.99-7.89 (m, 2H), 7.79 (d, 1H), 7.53-7.43 (m, 2H), 7.42-7.31 (m,1H), 3.87 (s, 2H), 3.53 (d, 1H), 3.20 (p, 2H), 3.07 (p, 2H), 2.78 (t,2H), 2.20 (s, 3H), 1.40 (s, 2H), 1.28 (q, 4H), 1.21-1.07 (m, 4H), 1.02(q, 2H), 0.84 (s, 6H). MS (ESI) m/c 850.1 (M+H)⁺.

1.71 Synthesis of6-[5-(1,3-benzothiazol-2-ylcarbamoyl)quinolin-3-yl]-3-{1-[(3-{2-[(2-carboxyethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (W2.71)

To a solution of Example 1.69.5 (140 mg) in N,N-dimethylformamide (10mL) was added tert-butyl acrylate (242 mg), and H₂O (0.3 mL), and themixture was stirred at room temperature over the weekend. The reactionmixture was diluted with dichloromethane and trifluoroacetic acid (10mL, 1:1) and stirred overnight. The mixture was concentrated, and theresidue was dissolved in N,N-dimethylformamide (4 mL) and purified byreverse-phase HPLC on a Gilson system (C18 column), eluting with 20-80%acetonitrile in water containing 0.1% trifluoroacetic acid, to give thetitle compound. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm 13.17 (s,2H), 9.69 (d, 1H), 9.37 (d, 1H), 8.30 (dd, 3H), 8.15 (dd, 1H), 8.04 (dd,1H), 7.99-7.88 (m, 2H), 7.79 (d, 1H), 7.53-7.40 (m, 2H), 7.34 (td, 1H),3.88 (s, 2H), 3.55 (t, 2H), 3.08 (dt, 4H), 2.62 (t, 2H), 2.21 (s, 3H),1.43 (s, 2H), 1.29 (q, 4H), 1.14 (s, 4H), 1.03 (q, 2H), 0.85 (s, 6H). MS(ESI) m/e 814.2 (M+H)⁺.

1.72 Synthesis of6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1³′1]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (W2.72) 1.72.1 Ethyl7-(5-bromo-6-(tert-butoxycarbonyl)pyridin-2-yl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate

The title compound was prepared by substituting ethyl5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate hydrochloride for1,2,3,4-tetrahydroisoquinoline-8-carboxylate hydrochloride in Example1.1.11. MS (ESI) m/e 451, 453 (M+H)⁺, 395, 397 (M-tert-butyl)⁺.

1.72.2 Ethyl7-(6-(tert-butoxycarbonyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate

The title compound was prepared by substituting Example 1.72.1 forExample 1.1.11 in Example 1.2.1. MS (ESI) m/e 499 (M+H)⁺, 443(M-tert-butyl)⁺. 529 (M+CH₃OH—H)⁻.

1.72.3 Ethyl7-(6-(tert-butoxycarbonyl)-5-(1-((3-(2-((tert-butoxycarbonyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)pyridin-2-yl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate

The title compound was prepared by substituting Example 1.72.2 forExample 1.2.1 and Example 1.55.11 for Example 1.13.3 in Example 1.13.4.MS (ESI) m/e 760 (M+H)⁺, 758 (M−H)⁻.

1.72.47-(6-(tert-butoxycarbonyl)-5-(1-((3-(2-((tert-butoxycarbonyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)pyridin-2-yl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylicAcid

The title compound was prepared by substituting Example 1.72.3 forExample 1.1.12 in Example 1.1.13. MS (ESI) m/e 760 (M+H)⁺, 758 (M−H)⁻.

1.72.5 Tert-butyl6-(1-(benzo[d]thiazol-2-ylcarbamoyl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl)-3-(1-((3-(2-((tert-butoxycarbonyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

The title compound was prepared by substituting Example 1.72.4 forExample 1.52.2 in Example 1.52.3. MS (ESI) m/e 892 (M+H)⁺, 890 (M−H)⁻.

1.72.63-(1-{[3-(2-aminoethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)-6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl]pyridine-2-carboxylicAcid

The title compound was prepared by substituting Example 1.72.5 forExample 1.1.16 in Example 1.1.17. MS (ESI) m/e 736 (M+H)⁺, 734 (M−H)⁻.

1.72.76-(1-(benzo[d]thiazol-2-ylcarbamoyl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl)-3-(1-((3-(2-((2-(((4-((tert-butyldiphenylsilyl)oxy)-2-methylbutan-2-yl)oxy)sulfonyl)ethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinicAcid

The title compound was prepared by substituting Example 1.72.6 forExample 1.2.7 in Example 1.2.8.

1.72.86-[1-(1,3-benzothiazol-2-ylcarbamoyl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid

The title compound was prepared by substituting Example 1.72.7 forExample 1.2.8 in Example 1.2.9. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆)δ ppm 8.36 (bs, 2H), 8.03 (bs, 1H), 7.99 (d, 1H), 7.76 (d, 1H), 7.64 (d,1H), 7.46 (t, 1H), 7.34 (s, 1H), 7.33 (t, 1H), 7.17 (d. H), 5.12 (s,2H), 4.28 (t, 2H), 4.11 (t, 2H), 3.86 (s, 2H), 3.56 (t, 2H), 3.24 (m,2H), 3.11 (m, 2H), 2.82 (t, 2H), 2.15 (s, 3H), 1.42 (s, 2H), 1.32 (q,4H), 1.17 (q, 4. H), 1.03 (m, 2H), 0.88 (s, 6H). MS (ESI) m/e 844(M+H)⁺, 842 (M−H)⁻.

1.73 Synthesis of8-(1,3-benzothiazol-2-ylcarbamoyl)-2-{6-carboxy-5-[1-({3-[2-({3-[1-(beta-D-glucopyranuronosyl)-1H-1,2,3-triazol-4-yl]propyl}amino)ethoxy]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridin-2-yl}-1,2,3,4-tetrahydroisoquinoline(W2.73)

To a solution of(2R,3R,4S,5S,6S)-2-azido-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (8.63 mg) in t-CH₃OH (2 mL) and water (1 mL) was addedExample 1.67.1 (20 mg), copper(II) sulfate pentahydrate (2.0 mg) andsodium ascorbate (5 mg). The mixture was stirred for 20 minutes at 100°C. under microwave conditions (Biotage Initiator). LiOH H₂O (50 mg) wasadded to the mixture, and stirring was continued overnight. The mixturewas neutralized with trifluoroacetic acid and purified by reverse-phaseHPLC on a Gilson system (C18 column), eluting with 20-80% acetonitrilein water containing 0.1% trifluoroacetic acid, to give the titlecompound. MS (APCI) m/e 987.3 (M+H)⁺.

1.74 Synthesis of6-[7-(1,3-benzothiazol-2-ylcarbamoyl)-1H-indol-2-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (W2.74) 1.74.1 methyl2-[5-{1-[(3-{2-[bis(tert-butoxycarbonyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-(tert-butoxycarbonyl)pyridin-2-yl]-1H-indole-7-carboxylate

Example 1.74.1 was prepared by substituting methyl2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole-7-carboxylatefor Example 1.2.1 and substituting Example 1.68.4 for Example 1.1.6 inExample 1.1.12. MS (ESI) m/e 866.3 (M−H)⁻.

1.74.22-(6-(tert-butoxycarbonyl)-5-(1-((3-(2-((tert-butoxycarbonyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)pyridin-2-yl)-1H-indole-7-carboxylicAcid

Example 1.74.2 was prepared by substituting Example 1.74.1 for Example1.1.12 in Example 1.1.13. MS (ESI) m/e 754.4 (M+H)⁺.

1.74.3 Tert-butyl6-(7-(benzo[d]thiazol-2-ylcarbamoyl)-1H-indol-2-yl)-3-(1-((3-(2-((tert-butoxycarbonyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

Example 1.74.3 was prepared by substituting Example 1.74.2 for Example1.1.13 in Example 1.1.14. MS (ESI) m/e 886.5 (M+H)⁺.

1.74.43-(1-((3-(2-aminoethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(7-(benzo[d]thiazol-2-ylcarbamoyl)-1H-indol-2-yl)picolinicAcid

Example 1.74.4 was prepared by substituting Example 1.74.3 for Example1.1.16 in Example 1.1.17. MS (ESI) m/e 730.2 (M+H)⁺.

1.74.56-[7-(1,3-benzothiazol-2-ylcarbamoyl)-1H-indol-2-yl]-3-[1-({3,5-dimethyl-7-[(2,2,7,7-tetramethyl-10,10-dioxido-3,3-diphenyl-4,9-dioxa-10l6-thia-13-aza-3-silapentadecan-5-yl)oxy]tricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylicAcid

Example 1.74.5 was prepared by substituting Example 1.74.4 for Example1.2.7 in Example 1.2.8. MS (ESI) m/e 1176.7 (M+H)⁺.

1.74.66-[7-(1,3-benzothiazol-2-ylcarbamoyl)-1H-indol-2-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid

Example 1.74.6 was prepared by substituting Example 1.74.5 for Example1.2.8 in Example 1.2.9. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm11.32 (d, 1H), 8.23 (dd, 1H), 8.18 (d, 1H), 7.93-7.82 (m, 3H), 7.71 (d,1H), 7.62 (s, 3H), 7.57-7.51 (m, 1H), 7.47 (s, 1H), 7.40 (d, 1). 7.35(t, 1H), 7.22 (t, 1H), 4.86 (t, 2H), 3.85 (s, 2), 3.47 (t, 2H), 3.08 (t,2H), 2.88 (p, 2H), 2.21 (s, 3H), 1.37 (s, 2H), 1.32-1.20 (m, 4H), 1.14(q, 4H), 1.07-0.94 (m, 2H), 0.84 (s, 6H). MS (ESI) m/e 838.2 (M+H)⁺.

1.75 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-6-[3-(methylamino)propyl]-3,4-dihydroisoquinolin-2(H)-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (W2.75) 1.75.1 Methyl 3-bromo-5-(bromomethyl)benzoate

Azobisisobutyronitrile (1.79 g) was added to methyl3-bromo-5-methylbenzoate (50 g) and N-bromosuccinimide (44.7 g) in 350mL acetonitrile, and the mixture was refluxed overnight. An additional11 g of N-bromosuccinimide and 0.5 g of azobisisobutyronitrile wasadded, and the refluxing was continued for 3 hours. The mixture wasconcentrated, taken up in 500 mL diethyl ether, and stirred for 30minutes. The mixture was filtered, and the resulting solution wasconcentrated. The crude product was chromatographed on silica gel using10% ethyl acetate in heptanes to give the title compound.

1.75.2 Methyl 3-bromo-5-(cyanomethyl)benzoate

Tetrabutylammonium cyanide (50 g) was added to Example 1.75.1 (67.1 g)in 300 mL acetonitrile, and the mixture was heated to 70° C. overnight.The mixture was cooled, poured into diethyl ether, and rinsed with waterand brine. The mixture was then concentrated and chromatographed onsilica gel using 2-20% ethyl acetate in heptanes to give the titlecompound.

1.75.3 Methyl 3-(2-aminoethyl)-5-bromobenzoate

Borane-THF complex (126 mL, 1M solution) was added to a solution ofExample 1.75.2 (16 g) in 200 mL tetrahydrofuran, and the mixture wasstirred overnight. The reaction was carefully quenched with methanol (50mL), and then concentrated to 50 mL volume. The mixture was taken up in120 mL methanol/120 mL 4M HCl/120 mL dioxane, and stirred overnight. Theorganics were removed under reduced pressure, and the residue wasextracted twice with diethyl ether. The extracts were discarded. Theorganic layer was basified with solid K₂CO₃, and then extracted withethyl acetate, and dichloromethane (2×). The extracts were combined,dried over Na₂SO₄, filtered and concentrated to give the title compound.

1.75.4 Methyl 3-bromo-5-(2-(2,2,2-trifluoroacetamido)ethyl)benzoate

Trifluoroacetic anhydride (9.52 mL) was added dropwise to a mixture ofExample 1.75.3 (14.5 g) and trimethylamine (11.74 mL) in 200 mLdichloromethane at 0° C. Upon addition the mixture was allowed to warmto room temperature and was stirred for three days. The mixture waspoured into diethyl ether, and washed with NaHCO₃ solution and brine.The mixture was concentrated and chromatographed on silica gel using5-30% ethyl acetate in heptanes to give the title compound.

1.75.5 Methyl6-bromo-2-(2,2,2-trifluoroacetyl)-1,2,3,4-tetrahydroisoquinoline-8-carboxylate

Sulfuric acid was added to Example 1.75.4 (10 g) until it went intosolution (40 mL), at which time paraformaldehyde (4.24 g) was added andthe mixture was stirred for 2 hours. The solution was then poured onto400 mL ice, and stirred 10 minutes. The mixture was extracted with ethylacetate (3×), and the combined extracts were washed with NaHCO₃ solutionand brine, and then concentrated The crude product was chromatographedon silica gel using 2-15% ethyl acetate in heptanes to give the titlecompound.

1.75.6 Methyl6-(3-((tert-butoxycarbonyl)methyl)amino)prop-1-yn-1-yl)-2-(2,2,2-trifluoroacetyl)-1,2,3,4-tetrahydroisoquinoline-8-carboxylate

A solution of Example 1.75.5 (5.1 g), tert-butylmethyl(prop-2-yn-1-yl)carbamate (2.71 g),bis(triphenylphosphine)palladium(I) dichloride (PdCl₂(PPh₃)₂, 0.49 g).CuI (0.106 g), and triethylamine (5.82 mL) was stirred in 50 mL dioxaneat 50° C. overnight. The mixture was concentrated and chromatographed onsilica gel using 10-50% ethyl acetate in heptanes to give the titlecompound.

1.75.7 Methyl6-(3-((tert-butoxycarbonyl)(methyl)amino)propyl)-2-(2,2,2-trifluoroacetyl)-1,2,3,4-tetrahydroisoquinoline-8-carboxylate

Example 1.75.6 (4.2 g), tetrahydrofuran (20 mL) and methanol (20.00 mL)were added to wet 20% Pd(OH)₂/C (3 g) in a 250 mL pressure bottle andshaken under a pressure of 50 psi and 50° C. for 12 hours. The solutionwas filtered and concentrated to give the title compound.

1.75.8 Methyl2-(5-bromo-6-(tert-butoxycarbonyl)pyridin-2-yl)-6-(3-((tert-butoxycarbonyl)(methyl)amino)propyl)-1,2,3,4-tetrahydroisoquinoline-8-carboxylate

Example 1.75.7 (4.22 g), and potassium carbonate (1.53 g) were stirredin 60 mL tetrahydrofuran, 25 mL methanol, and 10 mL water overnight. Themixture was concentrated and 60 mL N,N-dimethylformamide was added. Tothis was then added Example 1.1.9 (3.05 g) and triethylamine (5 mL), andthe reaction was stirred at 60° C. overnight. The mixture was cooled toroom temperature, poured into ethyl acetate (600 mL), washed with water(3×) and brine, dried over Na₂SO₄, filtered, and concentrated. Theresidue was chromatographed on silica gel using 5-50% ethyl acetate inheptanes to give the title compound. MS (ESI) m % e 618.2 (M+H)⁺.

1.75.9 Methyl6-(3-((tert-butoxycarbonyl)(methyl)amino)propyl)-2-(6-(tert-butoxycarbonyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)-1,2,3,4-tetrahydroisoquinoline-8-carboxylate

To a solution of Example 1.75.8 (3.7 g), triethylamine (2.50 mL) andPdCl₂(dppf)(([1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (1:1),0.29 g) in 25 mL acetonitrile was added4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1.74 mL), and the reactionmixture was heated to 75° C. for 5 hours, then stirred at 60° C.overnight. The mixture was concentrated and chromatographed on silicagel using 5-50% ethyl acetate in heptanes to give the title compound. MS(ESI) m/e 666.4 (M+H)⁺.

1.75.10 4-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylbutyl2-((2-((3-((4-iodo-5-methyl-1H-pyrazol-1-yl)methyl)-5,7-dimethyladamantan-1-yl)oxy)ethyl)amino)ethanesulfonate

Example 1.55.10 (2.39 g),4-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylbutyl ethenesulfonate (2.41g), and triethylamine (1.51 mL) were stirred in 30 mLN,N-dimethylformamide at 45° C. for 3 hours. The mixture was cooled andpoured into diethyl ether (400 mL), and the diethyl ether solution waswashed with water (3×) and brine, and concentrated. The crude productwas chromatographed on silica gel using 2-50% ethyl acetate in heptanes,with 1% added triethylamine to give the title compound. MS (ESI) m/e890.6 (M+H)⁺.

1.75.116-(6-(3-((tert-butoxycarbonyl)(methyl)amino)propyl)-8-(methoxycarbonyl)-3,4-dihydroisoquinolin-2(1H)-yl-3-(1-((3-(2-((2-((4-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylbutoxy)sulfonyl)ethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinicAcid

Example 1.75.9 (1.777 g), Example 1.75.10 (1.98 g),tris(dibenzylideneacetone)dipalladium(0) (0.102 g),1,3,5,7-tetramethyl-8-tetradecyl-2,4,6-trioxa-8-phosphaadamantane (0.918g), and potassium phosphate (1.889 g) were added to 25 mL dioxane/10 mLwater, and the solution was evacuated/filled with nitrogen severaltimes. The reaction was clear, and was stirred at 70° C. overnight. Themixture was cooled and poured into ethyl acetate (200 mL), and washedwith water and brine. The mixture was concentrated and chromatographedon silica gel using 5-50% ethyl acetate in heptanes, followed by 10%methanol in ethyl acetate with 1% triethylamine to give the titlecompound. MS (ESI) m/e 1301.4 (M+H)⁺.

1.75.126-(3-((tert-butoxycarbonyl)(methyl)amino)propyl)-2-(5-(1-((3-(2-((2-((4-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylbutoxy)sulfonyl)ethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-carboxypyridin-2-yl)-1,2,3,4-tetrahydroisoquinoline-8-carboxylicAcid

Example 1.75.11 (1.5 g) and LiOH—H₂O (0.096 g) were stirred in 15 mLtetrahydrofuran and 3 mL water at 45° C. for 10 days. The mixture waspoured into 200 mL ethyl acetate/20 mL NaH₂PO₄ solution, andconcentrated HCl solution was added until the pH reached 3. The layerswere separated, and the aqueous layer was extracted twice with ethylacetate. The combined organic layers were washed with brine andconcentrated. The residue was chromatographed on silica gel using 0-5%methanol in ethyl acetate to give the title compound. MS (ESI) m/e1287.3 (M+H)⁺.

1.75.136-(8-(benzo[d]thiazol-2-ylcarbamoyl)-6-(3-((tert-butoxycarbonyl)(methyl)amino)propyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-(2-((2-((4-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylbutoxy)sulfonyl)ethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinicAcid

The title compound was prepared as described in Example 1.2.6,substituting Example 1.2.5 with Example 1.75.12. MS (ESI) m/e 1419.5(M+H)⁺.

1.75.146-[8-(1,3-benzothiazol-2-ylcarbamoyl)-6-[3-(methylamino)propyl]-3,4-dihydriosoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-(2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1-]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylicAcid

The title compound was prepared as described in Example 1.2.9,substituting Example 1.2.8 with Example 1.75.13. ¹H NMR (400 MHz,dimethyl sulfoxide-d₆) δ ppm 12.90 (bs, 1H), 8.33 (m, 2H), 8.02 (d, 1H),7.78 (d, 1H), 7.66 (m, 1H), 7.47 (m, 3H), 7.35 (m, 3H), 7.25 (s, 2H),6.95 (d, 1H), 4.95 (s, 2H), 4.28 (t, 2H), 4.11 (t, 2H), 3.95 (m, 2H),3.20 (m, 2H), 3.08 (m, 2H), 2.96 (m, 2H), 2.89 (m, 2H), 2.78 (m, 2H),2.65 (m, 2H), 2.55 (t, 2H), 2.12 (s, 3H), 1.95 (m, 2H), 1.39 (s, 2H),1.25 (m, 6H), 1.12 (m, 6H), 0.93 (s, 3H), 0.85 (s, 6H). MS (ESI) m/e926.8 (M+H)⁺.

1.76 Synthesis of5-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]amino}-5-deoxy-D-arabinitol(W2.76) 1.76.1 Tert-butyl6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3,5-dimethyl-7-(2-((((4R,4′R,5R)-2,2,2′,2′-tetramethyl-[4,4′-bi(1,3-dioxolan)]-5-yl)methyl)amino)ethoxy)adamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

Example 1.2.7 (75 mg) and(4R,4′R,5S)-2,2,2′,2′-tetramethyl-[4,4′-bi(1,3-dioxolane)]-5-carbaldehyde(22 mg) were dissolved in dichloromethane (1 mL). Sodiumtriacetoxyborohydride (40 mg) was added, and the solution was stirredfor 16 hours at room temperature. The solution was concentrated underreduced pressure, and the material was purified by flash columnchromatography on silica gel, eluting with 5-10% methanol indichloromethane. The solvent was evaporated under reduced pressure toprovide the title compound. MS (ESI) m/e 1016 (M+H)⁺, 1014 (M−H)⁻.

1.76.25-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]amino}-5-deoxy-D-arabinitol

Example 1.76.1 (45 mg) was dissolved in trifluoroacetic acid (1 mL) andwater (0.2 mL). The solution was mixed at room temperature for fivedays. The solvents were removed under reduced pressure, and the materialwas taken up in methanol (2 mL). The material was purified byreverse-phase HPLC using 25-75% acetonitrile in water (w/0.1% TFA) over30 minutes on a Grace Reveleris equipped with a Luna column: C18(2), 100A, 250×30 mm. Product fractions were pooled, frozen, and lyophilized toyield the title compound as the bis trifluoroacetic acid salt. ¹H NMR(400 MHz, dimethyl sulfoxide-d₆) δ ppm 12.85 (bs, 2H), 8.31 (m, 1H),8.16 (m, 1H), 8.04 (d, 1H), 7.80 (d, 1H), 7.62 (d, 1H), 7.51-7.43 (m,3H), 7.37 (q, 2H), 7.29 (s, 1H), 6.69 (d, 1H), 4.96 (s, 2H), 4.04 (t,2H), 3.89 (m, 2H), 3.59 (m, 3H), 3.49 (m, 4H), 3.42 (dd, 2H), 3.22 (dd,2H), 3.06 (m, 2H), 3.02 (m, 4H), 2.10 (s, 3H), 1.43 (s, 2H), 1.30 (q,4H), 1.14 (t, 4H), 1.04 (q, 2H), 0.87 (s, 6H). MS (ESI) m/e 880 (M+H)⁺.878 (M−H)⁻.

1.77 Synthesis of1-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]amino}-1,2-dideoxy-D-arabino-hexitol(W2.77) 1.77.1 Tert-butyl6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3,5-dimethyl-7-(2-(((3R,4S,5R)-3,4,5,6-tetrahydroxyhexyl)amino)ethoxy)adamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

(4R,5S,6R)-6-(Hydroxymethyl)tetrahydro-2H-pyran-2,4,5-triol (15 mg) wasdissolved in dimethyl sulfoxide (0.5 mL). Example 1.2.7 (88 mg) wasadded, followed by sodium cyanoborohydride (27 mg). Acetic acid (82 mg)was added dropwise, and the solution was heated at 60° C. for 16 hours.The reaction was cooled, diluted with 1 mL of methanol, and purified byreverse-phase HPLC using 20-75% acetonitrile in water (w/0.1% TFA) over60 minutes on a Grace Reveleris equipped with a Luna column: C18(2), 100A, 150×30 mm. Product fractions were pooled, frozen, and lyophilized toyield the title compound as the bis trifluoroacetic acid salt. MS (ESI)m/e 950 (M+H)⁺. 948 (M−H)⁻.

1.77.21-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)-]dec-1-yl}oxy)ethyl]amino}-1,2-dideoxy-D-arabino-hexitol

Example 1.77.1 (39 mg) was dissolved in dichloromethane (0.5 mL).Trifluoroacetic acid (740 mg) was added, and the solution was stirred atroom temperature for 16 hours. The solvents were removed under reducedpressure. The residue was dissolved in N,N-dimethylformamide (0.5 mL)and 1 M aqueous sodium hydroxide (0.5 mL) was added. The solution wasstirred at room temperature for one hour. Trifluoroacetic acid (0.25 mL)was added, and the material was purified by reverse-phase HPLC using20-75% acetonitrile in water (w/0.1% TFA) over 60 minutes on a GraceReveleris equipped with a Luna column: C18(2), 100 A, 150×30 mm. Productfractions were pooled, frozen, and lyophilized to yield the titlecompound as the bis trifluoroacetic acid salt. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 12.86 (s, 1H), 12.74 (bs, 1H), 8.28 (bs, 1H), 8.20(bs, 1H), 8.04 (d, 1H), 7.80 (d, 1H), 7.62 (d, 1H), 7.51-7.43 (m, 3H),7.37 (q, 2H), 7.29 (s, 1H), 6.96 (d, 1H), 4.96 (s, 2H), 4.53 (bs, 3H),3.89 (t, 2H), 3.83 (s, 2H), 3.77 (d, 1H), 3.60 (dd, 2H), 3.56 (t, 2H),3.48 (m, 2H), 3.15 (d, 1H), 3.02 (m, 6H), 2.10 (s, 3H), 1.84 (m, 1H),1.69 (m, 1H), 1.43 (s, 2H), 1.31 (q, 4H), 1.14 (t, 4H), 1.05 (q, 2H),0.87 (s, 6H). MS (ESI) m/e 894 (M+H)⁺, 892 (M−H)⁻.

1.78 Synthesis of6-[4-(1,3-benzothiazol-2-ylcarbamoyl)isoquinolin-6-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (W2.78)

1.78.1 methyl6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoquinoline-4-carboxylate

To a solution of methyl 6-bromoisoquinoline-4-carboxylate (1.33 g) inN,N-dimethylformamide (30 mL) was added PdCl₂(dppf)-CH₂CH₂ adduct(([1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (1:1), 204mg), potassium acetate (1.48 g) and bis(pinacolato)diboron (1.92 g). Themixture was stirred at 60° C. overnight. The mixture was cooled to roomtemperature and used in the next reaction without further work up. MS(APCI) m/e 313.3 (M+H)⁺.

1.78.2 Methyl6-[5-{1-[(3-{2-[bis(tert-butoxycarbonyl}amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.17]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl)-6-(tert-butoxycarbonyl)pyridin-2-yl]isoquinoline-4-carboxylate

To a solution of the Example 1.68.4 (1.2 g) in 1,4-dioxane (20 mL) andwater (10 mL) was added Example 1.78.1 (517 mg),bis(triphenylphosphine)palladium(II) dichloride (58 mg), and CsF (752mg). The mixture was stirred at reflux overnight. LC/MS showed theexpected product as a major peak. The mixture was diluted with ethylacetate (200 mL), washed with water and brine, dried over anhydroussodium sulfate, filtered and concentrated. The residue was purified bysilica gel chromatography, eluting with 20% ethyl acetate indichloromethane to give the title compound. MS (ESI) m/e 880.8 (M+H)⁺.

1.7836-(6-(tert-butoxycarbonyl)-5-(1-((3-(2-((tert-butoxycarbonyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)pyridin-2-yl)isoquinoline-4-carboxylicAcid

To a solution of Example 1.78.2 (3.1 g) in tetrahydrofuran (20 mL),methanol (10 mL) and water (10 mL) was added LiOH H₂O (240 mg). Themixture was stirred at room temperature overnight. The mixture wasacidified with aqueous 2N HCl and diluted with ethyl acetate (400 mL).The organic layer was washed with water and brine and dried overanhydrous sodium sulfate. Filtration and evaporation of the solvent gavethe title compound. MS (ESI) m/e 766.4 (M+H)⁺.

1.78.43-(1-((3-(2-aminoethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(4-(benzo[d]thiazol-2-ylcarbamoyl)isoquinolin-6-yl)picolinicAcid

To a solution of Example 1.78.3 (1.2 g) in dichloromethane (20 mL) wasadded benzo[d]thiazol-2-amine (0.236 g),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (451 mg), and4-dimethylaminopyridine (288 mg), and the mixture was stirred at roomtemperature overnight. The reaction mixture was diluted with ethylacetate (500 mL), washed with water and brine, and dried over anhydroussodium sulfate. Filtration and evaporation of the solvent gave a residuethat was dissolved in dichloromethane and trifluoroacetic acid (10 mL,1:1) and stirred overnight. The mixture was concentrated, and theresidue was dissolved in N,N-dimethylformamide (4 mL) and purified byreverse-phase HPLC on a Gilson system (C18 column), eluting with 20-80%acetonitrile in water containing 0.1% trifluoroacetic acid, to give thetitle compound. MS (ESI) m/e 742.1 (M+H)⁺.

1.78.56-[4-(1,3-benzothiazol-2-ylcarbamoyl)isoquinolin-6-yl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)-]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid

To a solution of Example 1.78.4 (55 mg) in N,N-dimethylformamide (6 mL)was added 4-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylbutylethenesulfonate (34 mg). N,N-diisopropylthylamme (0.6 mL) and H₂O (0.6mL). The mixture was stirred at room temperature overnight. The reactionmixture was diluted with dichloromethane and trifluoroacetic acid (10mL, 1:1) and stirred overnight.

The mixture was concentrated, and the residue was dissolved inN,N-dimethylformamide (4 mL) and purified by reverse-phase HPLC on aGilson system (C18 column), eluting with 20-80% acetonitrile in watercontaining 0.1% trifluoroacetic acid, to give the title compound. ¹H NMR(400 MHz, dimethyl sulfoxide-d₆) δ ppm 13.25 (s, 2H), 9.58 (s, 1H), 9.06(s, 1H), 9.00 (s, 1H), 8.52 (dd, 1H), 8.42 (d, 1H), 8.35 (d, 2H), 8.26(d, 1H), 8.11-8.03 (m, 1H), 8.01 (d, 1H), 7.80 (d, 1H), 7.52-7.44 (m,2H), 7.41-7.28 (m, 1H), 3.89 (s, 2H), 3.55 (t, 2H), 3.22 (t, 2H), 3.09(s, 2H), 2.80 (t, 2H), 2.23 (s, 3H), 1.43 (s, 2H), 1.30 (q, 4H),1.23-1.11 (m, 4H), 1.04 (q, 2H), 0.86 (s, 6H). MS (ESI+) m/e 850.1(M+H)⁺.

1.79 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3-(2-{[3-hydroxy-2-(hydroxymethyl)propyl]amino}ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)-]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylicAcid (W2.79) 1.79.1 2,2-dimethyl-1,3-dioxane-5-carbaldehyde

To a stirred suspension of pyridinium chlorochromate (1.1 g) anddiatomaceous earth (10 g) in dichloromethane (10 mL) was added(2,2-dimethyl-1,3-dioxan-5-yl)methanol (0.5 g) as a solution indichloromethane (3 mL) dropwise. The mixture was stirred at roomtemperature for 2 hours. The suspension was filtered throughdiatomaceous earth and washed with ethyl acetate. The crude product wasfiltered through silica gel and concentrated to give the title compound.¹H NMR (501 MHz, chloroform-d) δ 9.89 (s, 1H), 4.28-4.17 (m, 4H),2.42-2.32 (m, 1H), 1.49 (s, 3H), 1.39 (s, 3H). MS (ESI) m/e 305.9(2M+NH₄)⁺.

1.79.2 Tert-butyl6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-(2-(((2,2-dimethyl-1,3-dioxan-5-yl)methyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

To a solution of Example 1.2.7 (100 mg) and Example 1.79.1 (20 mg) indichloromethane (1 mL) was added sodium triacetoxyborohydride (40 mg),and the mixture was stirred at room temperature for 2 hours. Thereaction was diluted with dichloromethane and washed with saturatedsodium bicarbonate solution. The aqueous layer was back extracted withdichloromethane. The combined organic layers were dried over sodiumsulfate, filtered and concentrated. Purification of the residue bysilica gel chromatography, eluting with 20%-100% ethyl acetate/ethanol(3:1) in heptane, provided the title compound. MS (ESI) m/e 930.3(M+H)⁺.

1.79.36-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-([3-(2-{[3-hydroxy-2-(hydroxymethyl)propyl]amino)ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylicAcid

Example 1.79.3 was prepared by substituting Example 1.79.2 for Example1.2.8 in Example 1.2.9. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm12.82 (s, 1H), 8.13 (s, 2H), 8.00 (dd, 1H), 7.76 (d, 1H), 7.59 (d, 1H),7.49-7.38 (m, 3H), 7.37-7.29 (m, 2H), 7.25 (s, 1H), 6.92 (d, 1H), 4.92(s, 4H), 3.85 (t, 2H), 3.79 (s, 2H), 3.53 (t, 2H), 3.47 (dd, 2H), 3.00(dt. 7H), 2.07 (s, 3H), 1.93 (p, 1H), 1.38 (s, 2H), 1.32-1.19 (m, 4H),1.16-0.91 (m, 6H), 0.83 (s, 7H). MS (ESI) m/e 834.3 (M+H)⁺.

1.80 Synthesis of1-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-yl)carbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]amino}-1,2-dideoxy-D-erythro-pentitol(W2.80)

The title compound was prepared by substituting(4S,5R)-tetrahydro-2H-pyran-2,4,5-triol for(4R,5S,6R)-6-(hydroxymethyl)tetrahydro-2H-pyran-2,4,5-triol and Example1.3.1 for Example 1.2.7 in Example 1.77.1. ¹H NMR (400 MHz, dimethylsulfoxide-d₆)(ppm 12.85 (bs, 1H), 12.72 (bs. 1H), 8.21 (bs, 2H), 8.04(d, 1H), 7.79 (d, 1H), 7.62 (d, 1H), 7.52-7.42 (m, 3H), 7.37 (q, 2H),7.29 (s, 1H), 6.95 (d, 1H), 4.96 (s, 2H), 3.89 (t, 2H), 3.83 (s, 2H),3.65 (m, 2H), 3.56 (m, 2H), 3.38 (m. 2H), 3.32 (m, 2H), 3.24 (m, 2H),3.03 (m, 5H), 2.10 (s, 3H), 1.89 (m, 1H), 1.67 (m, 1H), 1.44 (s, 2H),1.31 (q, 4H), 1.14 (t, 4H), 1.05 (q, 2H), 0.86 (s, 6H). MS (ESI) m/e 864(M+H)⁺, 862 (M−H)⁻.

1.81 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3,5-dimethyl-7-(2-{[(2S,3S)-2,3,4-trihydroxybutyl]amino}ethoxy)tricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylicAcid (W2.81) 1.81.1 Carbonic Acid Tert-butyl Ester(4S,5S)-5-hydroxymethyl-2,2-dimethyl-[1,3]dioxolan-4-ylmethyl Ester

((4S,5S)-2,2-Dimethyl-1,3-dioxolane-4,5-diyl)dimethanol (1000 mg) wasdissolved in N,N-dimethylfornamide (50 mL). Sodium hydride (60% inmineral oil, 259 mg) was added. The solution was mixed at roomtemperature for 15 minutes. Di-tert-butyl dicarbonate (1413 mg) wasadded slowly. The solution was mixed for 30 minutes, and the reactionwas quenched with saturated aqueous ammonium chloride solution. Thesolution was diluted with water (150 mL) and extracted twice using 70%ethyl acetate in heptanes. The organic portions were combined andextracted with water (100 mL), extracted with brine (50 mL), and driedon anhydrous sodium sulfate. The solution was concentrated under reducedpressure, and the material was purified by flash column chromatographyon silica gel eluting with 30% ethyl acetate in heptanes. The solventwas evaporated under reduced pressure to provide the title compound. MS(ESI) m/e 284 (M+Na)⁺.

1.81.2 Carbonic Acid Tert-butyl Ester(4,5R)-5-formyl-2,2-dimethyl-[1,3]dioxolan-4-ylmethyl Ester

Example 1.81.1 (528 mg) was dissolved in dichloromethane (20 mL).Dess-Martin periodinane (8% mg) was added, and the solution was stirredat room temperature for four hours. The solution was concentrated underreduced pressure, and the material was purified by flash columnchromatography on silica gel, eluting with 20%-50% ethyl acetate inheptanes. The solvent was evaporated under reduced pressure to providethe title compound.

1.81.3 Tert-butyl6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-(((1S,3s,5R,7S)-3-(2-((((4S,5S)-5-(((tert-butoxycarbonyl)oxy)methyl)-2,2-dimethyl-1,3-dioxolan-4-yl)methyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

The title compound was prepared by substituting Example 1.81.2 for(4R,4R,5S)-2,2,2′,2′-tetramethyl-[4,4′-bi(1,3-dioxolane)]-5-carbaldehydein Example 1.76.1.

1.81.46-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3,5-dimethyl-7-(2-{([(2S,3S)-2,3,4-trihydroxybutyl]amino}ethoxy)tricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylicAcid

The title compound was prepared by substituting Example 1.81.3 forExample 1.76.1 in Example 1.76.2. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 12.86 (bs, 2H), 8.28 (bs, 1H), 8.18 (bs, 1H), 8.04(d, 1H), 7.80 (d, 1H), 7.63 (d, 1H), 7.51-7.43 (m, 3H), 7.36 (q, 2H),7.29 (s, 1H), 6.96 (d, 1H), 4.96 (s, 2H), 3.89 (t, 2H), 3.83 (m, 3H),3.46 (m, 4H), 3.40 (m, 4H), 3.08-2.96 (m, 6H), 2.10 (s, 3H), 1.43 (s,2H), 1.30 (q, 4H), 1.14 (t, 4H), 1.04 (q, 2H), 0.87 (s, 6H). MS (ESI)m/e 850 (M+H)⁺, 848 (M−H)⁻.

1.82 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3-(2-{[(2S,3S,4R,5R,6R)-2,3,4,5,6,7-hexahydroxyheptyl]amino}ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylicAcid (W2.82)

The title compound was prepared by substituting(2R,3R,4S,5R,6R)-2,3,4,5,6,7-hexahydroxyheptanal for(4R,5S,6R)-6-(hydroxymethyl)tetrahydro-2H-pyran-2,4,5-triol and Example1.3.1 for Example 1.2.7 in Example 1.77.1. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 12.86 (bs, 1H), 8.34-8.08 (m, 2H), 8.05 (d, 1H),7.79 (d, 1H), 7.54-7.43 (m, 3H), 7.37 (m, 2H), 7.30 (s, 1H), 6.95 (d,1H), 4.96 (s, 2H), 3.93 (m, 2H), 3.90 (m, 4H), 3.83 (s, 2), 3.47 (m,4H), 3.41 (m, 4H), 3.18-3.08 (m, 7H), 3.03 (t, 2H), 2.12 (s, 3H), 1.46(s, 2H), 1.28 (q, 4H), 1.15 (t, 4H), 1.05 (q, 2H), 0.89 (s, 6H). MS(ESI) m/e 940 (M+H)⁺.

1.83 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[({3-[(1,3-dihydroxypropan-2-yl)amino]propyl)sulfonyl}amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (W2.83) 1.83.1 Tert-butyl6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-(2-(3-((1,3-dihydroxypropan-2-yl)amino)propylsulfonamido)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

To a cooled (ice bath) solution of Example 1.2.7 (31 mg) andN,N-diisopropylethylamine (60 μL) in dichloromethane (1 mL) was added3-chloropropane-1-sulfonyl chloride (5 μL). The mixture was stirred atroom temperature for 2 hours. The reaction was concentrated, dissolvedin N,N-dimethylformamide (1 mL), transferred to a 2 mL microwave tubeand 2-aminopropane-1,3-diol (70 mg) was added. The mixture was heated at130° C. under microwave conditions (Biotage Initiator) for 90 minutes.The reaction mixture was concentrated, and the residue was purified byreverse-phase HPLC using a Gilson system, eluting with 20-100%acetonitrile in water containing 0.1% v/v trifluoroacetic acid. Thedesired fractions were combined and freeze-dried to provide the titlecompound. MS (ESI) m/e 997.2 (M+H)⁺.

1.83.26-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[({3-[(1,3-dihydroxypropan-2-yl)amino]propyl}sulfonyl}amino]ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid

Example 1.83.2 was prepared by substituting Example 1.83.1 for Example1.2.8 in Example 1.2.9. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm12.84 (s, 1H), 8.40 (s, 2H), 8.05-7.98 (m, 1H), 7.77 (d, 1H), 7.60 (d,1H), 7.51-7.39 (m, 3H), 7.38-7.30 (m, 2H), 7.27 (s, 1H), 7.13 (t, 1H),6.93 (d, 1H), 4.94 (s, 2H), 3.61 (qd, 4H), 3.36 (t, 2H), 3.16-2.93 (m,10H), 2.08 (s, 3H), 20) (p, 2H), 1.38 (s, 2H), 1.25 (q, 4H), 1.15-0.92(m, 6H), 0.84 (s, 6H). MS (ESI) m/e 941.2 (M+H)⁺.

1.84 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[(3-{[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino}-3-oxopropyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (W2.84)

To a solution of tert-butyl3-(1-((3-(2-aminoethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinate(55 mg) in N,N-dimethylformamide (6 mL) was addedN-(1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)acylamide (73.4 mg).N,N-diisopropylethylamine (0.2 mL) and H₂O (0.2 mL). The mixture wasstirred at room temperature 4 days. LC/MS showed the expected product asa major peak. The reaction mixture was diluted with ethyl acetate (500mL), washed with water and brine, and dried over anhydrous sodiumsulfate. Filtration and evaporation of the solvent gave a residue thatwas dissolved in dichloromethane and trifluoroacetic acid (10 mL, 1:1)and stirred overnight. The mixture was concentrated, and the residue wasdissolved in N,N-dimethylformamide (8 mL) and purified by reverse-phaseHPLC on a Gilson system (C18 column), eluting with 20-80% acetonitrilein water containing 0.1% trifluoroacetic acid, to give the titlecompound. ¹H NMR (400 MHz, dimethylsulfonxide-d₆) δ ppm 12.84 (s, 1H),8.45 (s, 2H), 8.01 (d, 4H), 7.78 (d, 1H), 7.60 (d, 1H), 7.53-7.39 (m,3H), 7.39-7.30 (m, 2H), 7.27 (s, 1H), 6.94 (d, 1H), 4.94 (s, 2H), 4.14(s, 21), 3.87 (t, 2H), 3.81 (s, 2H), 3.52 (d, 4H), 3.19 (s, 3H),3.13-2.97 (m, SH), 2.75 (t, 2H), 2.08 (s, 3H), 1.42 (s, 2H), 1.29 (q,4H), 1.12 (s, 4H), 1.09-0.99 (m, 2H), 0.85 (s, 7H). MS (ESI) m/e 921.2(M+H)⁺.

1.85 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3-(2-{[(3S)-3,4-dihydroxybutyl]amino}ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylicAcid (W2.85)

To a solution of Example 1.2.7 (213 mg) in dichloromethane (2 mL) wasadded (S)-2-(2,2-dimethyl-1,3-dioxolan-4-yl)acetaldehyde (42 mg). Afterstirring at room temperature for 30 minutes, sodiumtriacetoxyborohydride (144 mg) was added. The reaction mixture wasstirred at room temperature overnight. Trifluoroacetic acid (2 mL) wasadded and stirring was continued overnight. The reaction mixture wasconcentrated, and the residue was purified by reverse-phase HPLC using aGilson system, eluting with 5-85% acetonitrile in water containing 0.1%v/v trifluoroacetic acid. The desired fractions were combined andfreeze-dried to provide the title compound. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 12.86 (s, 1H), 8.22 (d, 2H), 8.05-8.01 (m, 1H), 7.79(d, 1H), 7.61 (d, 1H), 7.53-7.41 (m, 3H), 7.36 (td, 2H), 7.28 (s, 1H),6.95 (d, 1H), 4.95 (s, 2H), 3.88 (t, 2H), 3.82 (s, 2H), 3.26-2.94 (m,7H), 2.10 (s, 3H), 1.84-1.75 (m, 1H), 1.52-1.63 (m, 1H), 1.45-1.23 (m,6H), 1.19-0.96 (m, 7H), 0.86 (s, 6H). MS (ESI) m/e 834.3 (M+H)⁺.

1.86 Synthesis of4-({-[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]amino}methyl)phenylbeta-D-glucopyranosiduronic Acid (W2.86)

To a solution of3-(1-((3-(2-aminoethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicacid (36 mg) in tetrahydrofuran (2 mL) and acetic acid (0.2 mL) wasadded(2S,3R,4S,5S,6S)-2-(4-formylphenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (21 mg) followed by MgSO₄ (60 mg). The mixture was stirred atroom temperature for 1 hour before the addition of MP-cyanoborohydride(Biotage, 153 mg, 2.49 mmol/g). The mixture was then stirred at roomtemperature for 3 hours. The mixture was filtered, and LiOH H₂O (20 mg)was added to the filtrate. The mixture was stirred at room temperaturefor 2 hours and then acidified with trifluoroacetic acid. The solutionwas purified by reverse-phase HPLC on a Gilson system (C18 column),eluting with 20-80% acetonitrile in water containing 0.1%trifluoroacetic acid, to give the title compound. MS (ESI) m/e 1028.3(M+H)⁺.

1.87 Synthesis of3-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]amino}propylbeta-D-glucopyranosiduronic Acid (W2.87) 1.87.1(2R,3R,5S,6S)-2-(3-hydroxypropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate

To a stirred solution of(2R,3R,5S,6S)-2-bromo-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (3.98 g) in toluene (60 mL) was added propane-1,3-diol (15.22g). The mixture was stirred at 75° C., and Ag₂CO₃ (5.52 g) was added inthree portions over a period of 3 hours. The mixture was stirred at roomtemperature overnight, after which the suspension was filtered. Thefiltrate was concentrated, and the residue was purified by silica gelchromatography eluting with 50% ethyl acetate in heptane to give thetitle compound. MS (ESI) m/e 409.9 (M+NH₄)⁺.

1.87.2(2S,3S,5R,6R)-2-(methoxycarbonyl)-6-(3-oxopropoxy)tetrahydro-2H-pyran-3,4,5-triylTriacetate

To a solution of dimethyl sulfoxide (0.5 mL) in dichloromethane (10 mL)at −78° C. was added oxalyl chloride (0.2 mL). The mixture was stirred20 minutes at −78° C., and a solution of Example 1.87.1 (393 mg) indichloromethane (10 mL) was added through a syringe. After 20 minutes,triethylaminc (1 mL) was added. The mixture was stirred for 30 minutes,and the temperature was allowed to rise to room temperature. Thereaction mixture was diluted with ethyl acetate (30 mL), washed withwater and brine, and dried over anhydrous sodium sulfate. Filtration andevaporation of the solvent gave the title compound, which was usedwithout further purification. MS (DCI) m/e 408.1 (M+NH₄)⁺.

1.87.33-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]amino}propylbeta-D-glucopyranosiduronic Acid

To a solution of Example 1.68.6 (171 mg) in dichloromethane (10 mL) wasadded Example 1.87.2 (90 mg), and NaBH(OAc)₃ (147 mg). The mixture wasstirred at room temperature overnight. The reaction mixture was dilutedwith ethyl acetate (200 mL), washed with 2% aqueous HCl solution, water,and brine, dried over anhydrous sodium sulfate, filtered andconcentrated. The residue was dissolved in tetrahydrofuran (6 mL),methanol (3 mL) and water (3 mL) and LiOH H₂O (100 mg) was added. Themixture was stirred at room temperature for 2 hours, acidified withtrifluoroacetic acid and concentrated under reduced pressure. Theresidue was dissolved in dimethyl sulfoxide/methanol (1:1, 12 mL) andpurified by reverse-phase HPLC on a Gilson system (C18 column), elutingwith 20-80% acetonitrile in water containing 0.1% trifluoroacetic acid)to give the title compound. ¹H NMR (400 MHz, dimethylsulfonxide-d₆) δppm 13.07 (s, 2H), 8.99 (s, 1H), 8.34 (dd, 1H), 8.29-8.11 (m, 5H),8.06-8.02 (m, 1H), 7.99 (d, 1H), 7.90 (d, 1H), 7.78 (d, 1H), 7.68 (dd,1H), 7.55-7.40 (m, 2H), 7.34 (td, 1H), 4.23 (d, 1H), 3.87 (s, 2H), 3.76(dt, 1H), 3.60 (d, 1H), 3.53 (dt, 3H), 3.29 (t, 1H), 3.15 (t, 1H),3.06-2.91 (m, 6H), 2.20 (s, 3H), 1.83 (p, 2H), 1.44 (s, 2H), 1.30 (q,4H), 1.14 (s, 4H), 1.03 (q, 2H), 0.85 (s, 7H). MS (ESI) m/e 975.2(M+H)⁺.

1.88 Synthesis of6-[4-(1,3-benzothiazol-2-ylcarbamoyl)-2-oxidoisoquinolin-6-yl]-3-[1-({3,5-dimethyl-7-[2-(methylamino)ethoxy]tricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylicAcid (W2.88) 1.88.1 methyl6-(6-(tert-butoxycarbonyl)-5-(1-((3-(2-((tert-butoxycarbonyl)(methyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)pyridin-2-yl)isoquinoline-4-carboxylate

To a solution of Example 1.78.1 (0.73 g) in 1,4-dioxane (20 mL) andwater (10 mL) was added tert-butyl3-(1-((3-(2-((tert-butoxycarbonyl)(methyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-chloropicolinate(1.5 g), bis(triphenylphosphine)palladium(II) dichloride (82 mg), andCsF (1.06 g), and the reaction was stirred at reflux overnight. Themixture was diluted with ethyl acetate (200 mL), washed with water andbrine, dried over anhydrous sodium sulfate, filtered, and concentrated.The residue was purified by silica gel chromatography, eluting with 20%ethyl acetate in heptane (1 L) to give the title compound. MS (ESI) m/e794.8 (M+H)⁺.

1.88.26-(6-(tert-butoxycarbonyl)-5-(1-((3-(2-((tert-butoxycarbonyl)(methyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)pyridin-2-yl)isoquinoline-4-carboxylicAcid

To a solution of Example 1.88.1 (300 mg) in tetrahydrofuran (6 mL),methanol (3 mL) and water (3 mL) was added LiOH H₂O (100 mg). Themixture was stirred at room temperature for 2 hours. The mixture wasacidified with aqueous 2N HCl solution, diluted with ethyl acetate (300mL), washed with water and brine, dried over anhydrous sodium sulfate,filtered and concentrated to give the title compound, which was usedwithout further purification. MS (ESI) m/e 781.2 (M+H)⁺.

1.88.3 Tert-butyl6-(4-(benzo[d]thiazol-2-ylcarbamoyl)isoquinolin-6-yl)-3-(1-((3-(2-((tert-butoxycarbonyl)(methyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

To a solution of Example 1.88.2 (350 mg) in dichloromethane (10 mL) wasadded benzo[d]thiazol-2-amine (67.5 mg),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (129 mg), and4-dimethylaminopyridine (82 mg). The mixture was stirred at roomtemperature overnight. The mixture was diluted with ethyl acetate (300mL), washed with water and brine, and dried over anhydrous sodiumsulfate. Filtration and evaporation of the solvent gave a residue, whichwas purified by silica gel chromatography, eluting with 5% methanol indichloromethane, to give the title compound. MS (APCI) m/e 912.3 (M+H)⁺.

1.88.44-(benzo[d]thiazol-2-ylcarbamoyl)-6-(6-carboxy-5-(1-((3,5-dimethyl-7-(2-(methylamino)ethoxy)adamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)pyridin-2-yl)isoquinoline2-oxide

To a solution of Example 1.88.3 (100 mg) in dichloromethane (6 mL) wasadded m-chloroperoxybenzoic acid (19 mg). The mixture was stirred atroom temperature for 4 hours. The mixture was diluted with ethyl acetate(200 mL), washed with saturated aqueous NaHCO₃ solution, water, andbrine, and dried over anhydrous sodium sulfate. Filtration andevaporation of the solvent gave a residue that was dissolved indichloromethane/trifluoroacetic acid (10 mL, 1:1) and stirred at roomtemperature overnight. The solvents were evaporated, and the residue waspurified by reverse-phase HPLC on a Gilson system (C18 column), elutingwith 20-80% acetonitrile in water containing 0.1% trifluoroacetic acid,to give the title compound. ¹H NMR (501 MHz, dimethyl sulfoxide-d₆) δppm 13.32 (s, 2H), 9.21 (d, 1H), 8.71 (d, 1H), 8.49 (dd, 1H), 8.36-8.19(m, 4H), 8.12 (dd, 1H), 8.07 (d, 1H), 7.96 (dd, 1H), 7.82 (d, 1H),7.56-7.46 (m, 3H), 7.42-7.35 (m, 1H), 3.90 (d, 3H), 3.56 (td, 3H), 3.02(p, 3H), 2.55 (t, 4H), 2.29-2.19 (m, 4H), 1.45 (d, 3H), 1.37-1.26 (m,5H), 1.16 (d, 6H), 1.10-1.01 (m, 3H), 0.88 (d, 8H). MS (ESI) m/e 772.1(M+H)⁺.

1.89 Synthesis of6-{8-[(1,3-benzothiazol-2-yl)carbamoyl]-3,4-dihydroisoquinolin-2(1H)-yl}-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]acetamido}tricyclo[3.3.1.1^(3,7)]decan-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (W2.89) 1.89.11-((3-bromo-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazole

To a cooled (−30° C.) solution of Example 1.1.3 (500 mg) intetrahydrofuran (30 mL) was added n-butyllithium (9.67 mL), and themixture was stirred at −30° C. for 2 hours. Methyl iodide (1.934 mL) wasadded dropwise at −30° C. After completion of the addition, the mixturewas stirred at −30° C. for additional 2 hours. 1N aqueous HCl in icewater was added slowly, such that the temperature was maintained below0° C., until the pH reached 6. The mixture was stirred at roomtemperature for 10 minutes, and diluted with ice-water (10 mL) and ethylacetate (20 mL). The layers were separated, and the aqueous layer wasextracted twice with ethyl acetate. The combined organic phases werewashed with brine, dried over MgSO₄, filtered and concentrated. Theresidue was purified by flash silica gel chromatography, eluting with15/1 to 10/1petroleumeum/ethyl acetate, to give the title compound. MS(LC-MS) m/e 337,339 (M+H)⁺.

1.89.21-(3,5-dimethyl-7-((5-methyl-1H-pyrazol-1-yl)methyl)adamantan-1-yl)urea

Example 1.89.1 (2.7 g) and urea (4.81 g) was mixed and stirred at 140°C. for 16 hours.

The mixture was cooled to room temperature and suspended in methanol(200 mL×2). The insoluble material was removed by filtration. Thefiltrate was concentrated to give the title compound. MS (LC-MS) me317.3 (M+H)⁺.

1.89.33,5-dimethyl-7-((5-methyl-1H-pyrazol-1-yl)methyl)adamantan-1-amine

To a solution of Example 1.40.2 (2.53 g) in 20% ethanol in water (20 mL)was added sodium hydroxide (12.79 g). The mixture was stirred at 120° C.for 16 hours and at 140° C. for another 16 hours. 6N Aqueous HCl wasadded until pH 6. The mixture was concentrated, and the residue wassuspended in methanol (200 mL). The insoluble material was filtered off.The filtrate was concentrated to give the title compound as an HCl salt.MS (LC-MS) m/e 273.9 (M+H)⁺.

1.89.4 Tert-butyl(2-((3,5-dimethyl-7-((S-methyl-1H-pyrazol-1-yl)methyl)adamantan-1-yl)amino)-2-oxoethyl)carbamate

To a solution of Example 1.89.3 (2.16 g) in N,N-dimethylformamide (100mL) was added triethylamine (3.30 mL),2-((tert-butoxycarbonyl)amino)acetic acid (1.799 g) and1-[bis(dimethylamino)methylene]-1H-12,3-triazolo[4,5-b]pyridinium 3-oxidhexafluorophosphate (3.90 g). The mixture was stirred at roomtemperature for 2 hours. Water (40 mL) was added, and the mixture wasextracted with ethyl acetate (70 mL×2). The combined organic phases werewashed with brine, dried over sodium sulfate, filtered and concentrated.The residue was purified by silica gel chromatography, eluting with 3/1to 2/1 petroleum/ethyl acetate, to give the title compound. MS (LC-MS)m/e 430.8 (M+H)⁺.

1.89.5 Tert-butyl(2-((3-((4-iodo-5-methyl-1H-pyrazol-1-yl)methyl)-5,7-dimethyladamantan-1-yl)amino)-2-oxoethyl)carbamate

To an ambient solution of Example 1.89.4 (1.7 g) inN,N-dimethylformamide (20 mL) was added NIS (N-iodosuccinimide, 1.066 g)in portions, and the mixture was stirred at room temperature for 16hours. Ice-water (10 mL) and saturated aqueous Na₂S₂O₃ solution (10 mL)were added. The mixture was extracted with ethyl acetate (30 mL×2). Thecombined organic phases were washed with brine, dried over sodiumsulfate, filtered and concentrated. The residue was purified by silicagel chromatography, eluting with 3/1 to 2/1 petroleum/ethyl acetate, togive the title compound. MS (LC-MS) m/e 556.6 (M+H)⁺.

1.89.6 Methyl2-(5-bromo-6-(tert-butoxycarbonyl)pyridin-2-yl)-1,2,3,4-tetrahydroisoquinoline-8-carboxylate

To a solution of methyl 1,2,3,4-tetrahydroisoquinoline-8-carboxylatehydrochloride (12.37 g) and Example 1.1.10 (15 g) in dimethyl sulfoxide(100 mL) was added N,N-diisopropylethylamine (12 mL), and the mixturewas stirred at 50° C. for 24 hours. The mixture was then diluted withethyl acetate (500 mL) and washed with water and brine. The organiclayer was dried over sodium sulfate, filtered and concentrated underreduced pressure. The residue was purified by silica gel chromatography,eluting with 20% ethyl acetate in hexane, to give the title compound. MS(ESI) m/e 448.4 (M+H)⁺.

1.89.7 Methyl2-(6-(tert-butoxycarbonyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)-1,2,3,4-tetrahydroisoquinoline-8-carboxylate

To a solution of Example 1.89.6 (2.25 g) and[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II)(205 mg) inacetonitrile (30 mL) was added triethylamine (3 mL) and pinacolborane (2mL), and the mixture was stirred at reflux for 3 hours. The mixture wasdiluted with ethyl acetate (200 mL) and washed with water and brine. Theorganic layer was dried over sodium sulfate, filtered and concentratedunder reduced pressure. Purification of the residue by flashchromatography, eluting with 20% ethyl acetate in hexane, provided thetitle compound.

1.89.8 Methyl2-(6-(tert-butoxycarbonyl)-5-(1-((3-(2-((tert-butoxycarbonyl)amino)acetamido)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)pyridin-2-yl)-1,2,3,4-tetrahydroisoquinoline-8-carboxylate

The title compound was prepared using the procedure in Example 1.2.2,substituting Example 1.1.6 with Example 1.89.5. MS (ESI) m/e 797.4(M+H)⁺.

1.89.92-(6-(tert-butoxycarbonyl)-5-(1-((3-(2-((tert-butoxycarbonyl)amino)acetamido)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)pyridin-2-yl)-1,2,3,4-tetrahydroisoquinoline-8-carboxylicAcid

The title compound was prepared using the procedure in Example 1.2.5,substituting Example 1.2.4 with Example 1.89.8. MS (ESI) m/e 783.4(M+H)⁺.

1.89.10 Tert-butyl6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-(2-((tert-butoxycarbonyl)amino)acetamido)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

The title compound was prepared using the procedure in Example 1.2.6,substituting Example 1.2.5 with Example 1.89.9. MS (ESI) m/e 915.3(M+H)⁺.

1.89.113-(1-{[3-(2-aminoacetamido)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]decan-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)-6-{8-[(1,3-benzothiazol-2-yl)carbamoyl]-3,4-dihydroisoquinolin-2(1H)-yl}pyridine-2-carboxylicAcid

The title compound was prepared using the procedure in Example 1.2.9,substituting Example 1.2.8 with Example 1.89.10. ¹H NMR (400 MHz,dimethyl sulfoxide-d₆) δ 12.82 (s, 1H), 8.00 (dd, 1H), 7.90-7.79 (m,4H), 7.76 (d, 1H), 7.59 (dd, 1H), 7.49-7.38 (m, 3H), 7.37-7.29 (m, 2H),7.25 (s, 1H), 6.92 (d, 1H), 4.92 (s, 2H), 3.85 (t, 2H), 3.77 (s, 2H),3.40 (q, 2H), 2.98 (t, 2H), 2.07 (s, 3H), 1.63 (s, 2H), 1.57-1.38 (m,4H), 1.15-0.93 (m, 6H), 0.80 (s, 6H). MS (ESI) m/e 759.2 (M+H)⁺.

1.89.126-{8-[(1,3-benzothiazol-2-yl)carbamoyl]-3,4-dihydroisoquinolin-2(1H)-yl}-3-(1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]acetamido}tricyclo[3.3.1.1^(3,7)]decan-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylicAcid

To a solution of Example 1.89.11 (102 mg) in N,N-dimethylformamide (6mL) was added 4-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylbutylethenesulfonate (60 mg),and the mixture was stirred at room temperatureover a weekend. The mixture was diluted with ethyl acetate (300 mL),washed with water and brine, and dried over anhydrous sodium sulfate.Filtration and evaporation of the solvent gave a residue that wasdissolved in dichloromethane/trifluoroacetic acid (10 mL, 1:1) andstirred at room temperature overnight. The solvents were evaporated, andthe residue was purified by reverse-phase HPLC on a Gilson system (C18column), eluting with 20-80% acetonitrile in water containing 0.1%trifluoroacetic acid, to give the title compound. ¹H NMR (501 MHz,dimethyl sulfoxide-d₆) δ 12.83 (s, 1H), 8.57 (s, 2H), 8.02 (d, 1H), 7.95(s, 1H), 7.77 (d, 1H), 7.60 (d, 1H), 7.52-7.37 (m, 3H), 7.39-7.29 (m,2H), 7.26 (s, 1H), 6.94 (d, 1H), 4.94 (s, 2H), 3.87 (t, 2H), 3.79 (s,2H), 3.16 (q, 2H), 2.99 (t, 2H), 2.77 (t, 2H), 2.08 (s, 3H), 1.64 (s,2H), 1.55 (d, 2H), 1.45 (d, 2H), 1.21-0.95 (m, 6H), 0.82 (s, 6H). MS(ESI) m/e 867.2 (M+H)⁺.

1.90 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3,5-dimethyl-7-({2-[(2-sulfoethyl)amino]ethyl}sulfanyl)tricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylicAcid (W2.90) 1.90.13-((1H-pyrazol-1-yl)methyl)-5,7-dimethyladamantane-1-thiol

A mixture of Example 1.1.3 (2.8 g) and thiourea (15.82 g) in 33% (w/w)HBr in acetic acid (50 mL) was stirred at 110° C. for 16 hours and wasconcentrated under reduced pressure to give a residue. The residue wasdissolved in 20% ethanol in water (v/v: 200 mL), and sodium hydroxide(19.06 g) was added. The resulting solution was stirred at roomtemperature for 16 hours and was concentrated. The residue was dissolvedin water (60 mL), and acidified with 6 N aqueous HCl to pH 5-pH 6. Themixture was extracted with ethyl acetate (200 mL×2). The combinedorganic layers were washed with brine, dried over MgSO₄, filtered andconcentrated to give the title compound. MS (ESI) m/e 319.1 (M+H)⁺.

1.90.22-((-3-((1H-pyrazol-1-yl)methyl)-5,7-dimethyladamantan-1-yl)thio)ethanol

To a solution of Example 1.90.1 (3.3 g) in ethanol (120 mL) was addedsodium ethoxide (2.437 g). The mixture was stirred for 10 minutes, and2-chloroethanol (1.80 mL) was added dropwise. The mixture was stirred atroom temperature for 6 hours and was neutralized with 1 N aqueous HCl topH 7. The mixture was concentrated, and the residue was extracted withethyl acetate (200 mL×2). The combined organic layers were washed withbrine, dried over MgSO₄, filtered and concentrated. The residue waspurified by column chromatography on silica gel, eluting with petroleumether/ethyl acetate from 6/1 to 2/1, to give the title compound. MS(ESI) m/e 321.2 (M+H)⁺.

1.90.32-((-3,5-dimethyl-7-((5-methyl-1H-pyrazol-1-yl)methyl)adamantan-1-yl)thio)ethanol

To a solution of Example 1.90.2 (2.3 g) in tetrahydrofuran (60 mL) wasadded n-butyllithium (14.35 mL, 2M in hexane) at −20° C. dropwise undernitrogen. The mixture was stirred at this temperature for 2 hours.Methyl iodide (4.49 mL) was added to the resulting mixture at −20° C.,and the mixture was stirred at −20° C. for 2 hours. The reaction wasquenched by the dropwise addition of saturated aqueous NH₄Cl solution at−20° C. The resulting mixture was stirred for 10 minutes and acidifiedwith 1 N aqueous HCl to pH 5. The mixture was extracted with ethylacetate twice. The combined organic layers were washed with brine, driedover MgSO₄, filtered and concentrated to give the title compound. MS(ESI) m/e 335.3 (M+H)⁺.

1.90.42-((-3-((4-iodo-5-methyl-1H-pyrazol-1-yl)methyl)-5,7-dimethyladamantan-1-yl)thio)ethanol

To a solution of Example 1.90.3 (3.65 g) in N,N-dimethylformamide (90mL) was added N-iodosuccinimide (3.68 g). The mixture was stirred atroom temperature for 16 hours. The reaction was quenched by the additionof ice-water (8 mL) and saturated aqueous NaS₂O₃ solution (8 mL). Themixture was stirred for an additional 10 minutes and was extracted withethyl acetate (30 mL×2). The combined organic layers were washed withbrine, dried over MgSO₄, filtered and concentrated under reducedpressure. The residue was purified by silica gel chromatography, elutingwith petroleum ether/ethyl acetate (6/1 to 3/1), to give the titlecompound. MS (ESI) m/e 461.2 (M+H)⁺.

1.90.5 Di-tert-butyl[2-({3-[(4-iodo-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]decan-1-yl}sulfanyl)ethyl]-2-imidodicarbonate

To a cold solution (0° C. bath) of Example 1.90.4 (3 g) indichloromethane (100 mL) was added triethylamine (1.181 mL) and mesylchloride (0.559 mL). The mixture was stirred at room temperature for 4hours, and the reaction was quenched by the addition of ice-water (30mL). The mixture was stirred for an additional 10 minutes and wasextracted with dichloromethane (50 mL×2). The combined organic layerswere washed with brine, dried over MgSO₄, filtered and concentratedunder reduced pressure. The residue was dissolved in acetonitrile (100mL) and NH(Boc)₂ (1.695 g) and Cs₂CO₃ (4.24 g) were added. The mixturewas stirred at 85° C. for 16 hours, and the reaction was quenched by theaddition of water (20 mL). The mixture was stirred for 10 minutes andwas extracted with ethyl acetate (40 mL×2). The combined organic layerswere washed with brine, dried over MgSO₄, filtered and concentrated. Theresidue was purified by silica gel chromatography, eluting withpetroleum ether/ethyl acetate from 10/1 to 6/1, to give the titlecompound. MS (ESI) m/e 660.1 (M+H)⁺.

1.90.6 methyl2-[5-(1-{[3-({2-[bis(tert-butoxycarbonyl)amino]ethyl}sulfanyl)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]decan-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)-6-(tert-butoxycarbonyl)pyridin-2-yl]-1,2,3,4-tetrahydroisoquinoline-8-carboxylate

The title compound was prepared using the procedure in Example 1.2.2,replacing Example 1.1.6 with Example 1.90.5. MS (ESI) m/e 900.2 (M+H)⁺.

190.7A2-(6-(tert-butoxycarbonyl)-5-(1-((3-((2-((tert-butoxycarbonyl)amino)ethyl)thio)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)pyridin-2-yl)-1,2,3,4-tetrahydroisoquinoline-8-carboxylicAcid

The title compound was prepared as described in Example 1.2.5, replacingExample 1.2.4 with Example 1.90.6. MS (ESI) m/e 786.2 (M+H)⁺.

190.7B tert-butyl6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-((2-((tert-butoxycarbonyl)amino)ethyl)thio)-5,7-dimethyladamantan-1-y)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

The title compound was prepared as described in Example 1.2.6, replacingExample 1.2.5 with Example 1.90.7A. MS (ESI) m/e 918.8 (M+H)⁺.

1.90.8 Tert-butyl3-(1-((3-((2-aminoethyl)thio)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinate

To a solution of Example 1.90.7B (510 mg) in dichloromethane (5 mL) wasadded trifluoroacetic acid (5 mL,) and the reaction was stirred at roomtemperature for 30 minutes. The reaction was quenched by the addition ofsaturated aqueous sodium bicarbonate solution and extracted withdichloromethane thrice. The combined organics were dried with anhydroussodium sulfate, filtered and concentrated under reduced pressure. Theresidue was purified by reverse-phase HPLC on a Gilson system (C18column), eluting with 20-80% acetonitrile in water containing 0.1%trifluoroacetic acid, to give the title product. MS (ESI) m/e 818.1(M+H)⁺.

1.90.93-(1-((3-((2-aminoethyl)thio)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid

Example 1.90.9 was isolated during the preparation of Example 1.90.8. MS(ESI) 762.2 (M+H)⁺.

1.90.10 Tert-butyl6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-((2-((2-((4-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylbutoxy)sulfonyl)ethyl)amino)ethyl)thio)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

Example 1.90.8 (235 mg) and4-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylbutyl ethenesulfonate (150mg) were dissolved in dichloromethane (1 mL), N,N-diisopropylethylamine(140 μL) was added, and the mixture was stirred at room temperature forsix days. The reaction was directly purified by silica gelchromatography, eluting with a gradient of 0.5-3.0% methanol indichloromethane, to give the title compound.

1.90.116-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3,5-dimethyl-7-((2-((2-sulfoethyl)amino)ethyl)thio)adamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinicAcid

The title compound was prepared by substituting Example 1.90.10 forExample 1.2.8 in Example 1.2.9. ¹H NMR (500 MHz, dimethyl sulfoxide-d₆)δ ppm 8.39 (br s, 2H), 8.03 (d, 1H), 7.79 (d, 1H), 7.62 (d, 1H), 7.51(d, 1H),7.47 (ddd, 1H), 7.43 (d, 1H), 7.37 (d, 1H), 7.35 (ddd, 1H), 7.30(s, 1H), 6.96 (d, 1H), 4.96 (s, 2H), 3.89 (t, 2H), 3.81 (s, 2H), 3.22(m, 2H), 3.06 (br in, 2H), 3.01 (t, 2H), 2.79 (t, 2H), 2.74 (m, 2H),2.10 (s, 3H), 1.51 (s, 2H), 1.37 (m, 4H), 1.15 (m, 4H), 1.05 (m, 2H),0.83 (s, 6H). MS (ESI) m/e 870.1 (M+H)⁺.

1.91 Synthesis of6-[8-[(1,3-benzothiazol-2-yl)carbamoyl]-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3,5-dimethyl-7-{(3-[(2-sulfoethyl)amino]propyl}tricyclo[3.3.1.1^(3,7)]decan-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (W2.91) 1.91.11-((3-allyl-5,7-dimethyladamantan-1-yl)methyl)-1H-pyrazole

To a solution of Example 1.1.3 (0.825 g. 2.55 mmol) in toluene (5 mL)was added N, N′-azoisobutyronitrile (AIBN, 0.419 g, 2.55 mmol) andallyltributylstannane (2.039 ml, 6.38 mmol). The mixture was purged withN₂ stream for 15 minutes, heated at 80° C. for 8 hours and concentrated.The residue was purified by flash chromatography, eluting with 5% ethylacetate in petroleum ether to provide the title compound. MS (ESI) m/e285.2 (M+H)⁺.

1.91.21-((3-allyl-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazole

To a solution of Example 1.91.1 (200 mg, 0.703 mmol) in tetrahydrofuran(5 ml) at −78° C. under N₂ was added n-butyllithium (2.81 mL, 7.03mmol). The mixture was stirred for 2 hours while the temperatureincreased to −20° C., and then it was stirred at −20° C. for 1 hour.Iodomethane (0.659 ml, 10.55 mmol) was added and the resulting mixturewas stirred for 0.5 hours at −20° C. The reaction was quenched withsaturated NH₄Cl and extracted with ethyl acetate twice. The combinedorganic layer was washed with brine and concentrated to give the titlecompound. MS (ESI) m/e 299.2 (M+H)⁺.

1.91.33-(3,5-dimethyl-7-((5-methyl-1H-pyrazol-1-yl)methyl)adamantan-1-yl)propan-1-ol

Under nitrogen atmosphere, a solution of Example 1.91.2 (2.175 g, 7.29mmol) in anhydrous tetrahydrofuran (42.5 mL) was cooled to 0° C. BH₃.THF(15.30 mL, 15.30 mmol) was added dropwise. The reaction mixture wasstirred at room temperature for 2 hours and cooled to 0° C. To thereaction mixture was added 10 N aqueous NaOH (5.03 mL, 50.3 mmol)dropwise, followed by 30 percent H₂O₂ (16.52 mL, 146 mmol) watersolution. The resulting mixture was warmed to room temperature andstirred for 90 minutes. The reaction was quenched with 10 percenthydrochloric acid (35 mL). The organic layer was separated and theaqueous layer was extracted with ethyl acetate (2×60 mL). The combinedorganic layers were washed with brine (3×60 mL) and cooled in an icebath. A saturated aqueous solution of sodium sulfite (15 mL) wascarefully added and the mixture was stirred for a few minutes. Theorganic layer was dried over sodium sulfate, filtered, and concentratedin vacuo. The residue was purified by flash chromatography, eluting withpetroleum ether/ethyl acetate (3:1 to 1:1) to provide the titlecompound. MS (ESI) m/e 317.3 (M+H)⁺.

1.91.43-(3-((4-iodo-5-methyl-1H-pyrazol-1-yl)methyl)-5,7-dimethyladamantan-1-yl)propan-1-ol

A mixture of Example 1.91.3 (1.19 g, 3.76 mmol) and 1-iodopyrrolidine-2,5-dione (1.015 g. 4.51 mmol) in N,N-dimethylformamide (7.5 mL) wasstirred for 16 hours at room temperature. The reaction was quenched withsaturated Na₂SO₃ The mixture was diluted with ethyl acetate and washedwith saturated Na₂SO₃, saturated Na₂CO₃, water and brine. The organiclayer was dried over anhydrous Na₂SO₄, filtered, and concentrated. Theresidue was purified by flash chromatography, eluting with petroleumether/ethyl acetate (3:1 to 1:1) to provide the title compound. MS (ESI)m/e 443.1 (M+H)⁺.

1.91.53-(3-((4-iodo-5-methyl-1H-pyrazol-1-yl)methyl)-5,7-dimethyladamantan-1-yl)propylMethanesulfonate

To a solution of Example 1.91.4 (1.55 g, 3.50 mmol) in CH₂Cl₂ (20 mL) at0° C. were added (CH₃CH₂)₃N (0.693 mL, 4.98 mmol) and mesyl chloride(0.374 mL, 4.80 mmol) slowly. The mixture was stirred for 3.5 hours at20° C., and diluted with CH₂C2, washed with saturated NH₄Cl. NaHCO₃ andbrine. The organic layer was dried over Na₂SO₄, filtered, andconcentrated to provide the title compound. MS (ESI) m/e 521.1 (M+H)⁺.

1.91.6 di-tert-butyl(3-{3-[(4-iodo-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]decan-1-yl}propyl)-2-imidodicarbonate

To a solution of Example 1.91.5 (1.92 g, 3.69 mmol) in CH3CN (40 ml) at20° C. was added di-tert-butyl iminodicarbonate (0.962 g. 4.43 mmol) andCs₂CO₃ (2.404 g. 7.38 mmol). The mixture was stirred for 16 hours at 80°C., and was diluted with ethyl acetate, and was washed with water andbrine. The organic layer was dried over Na₂SO₄, filtered, andconcentrated. The residue was purified by flash chromatography, elutingwith petroleum ether/ethyl acetate (10:1) to provide the title compound.MS (ESI) m/e 642.3 (M+H)⁺.

1.91.7 methyl2-[5-{1-[(3-{3-[bis(tert-butoxycarbonyl}amino]propyl-5,7-dimethyltricyclo[3.3.1.1^(3,7)]decan-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}-6-(tert-butoxycarbonyl)pyridin-2-yl]-1,2,3,4-tetrahydroisoquinoline-8-carboxylate

The title compound was prepared using the procedure in Example 1.2.2,replacing Example 1.1.6 with Example 1.91.6. MS (ESI) m/e 882.2 (M+H)⁺.

1.91.82-[6-(tert-butoxycarbonyl)-5-{1-[(3-{3-[(tert-butoxycarbonyl)amino]propyl}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]decan-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridin-2-yl]-1,2,3,4-tetrahydroisoquinoline-8-carboxylicAcid

The title compound was prepared using the procedure in Example 1.2.5,replacing Example 1.2.4 with Example 1.91.7. MS (ESI) m/e 768.4 (M+H)⁺.

1.91.9 Tert-butyl6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-(3-((tert-butoxycarbonyl)amino)propyl)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

The title compound was prepared using the procedure in Example 1.2.6,replacing Example 1.2.5 with Example 1.91.8. MS (ESI) m/e 901.1 (M+H)⁺.

1.91.10 Tert-butyl3-(1-((3-(3-aminopropyl)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinate

To a solution of Example 1.91.9 (500 mg) in dichloromethane (5 mL) wasadded trifluoroacetic acid (5 mL) and the reaction was stirred at roomtemperature for 30 minutes. The reaction was quenched by the addition ofsaturated aqueous sodium bicarbonate solution and extracted withdichloromethane thrice. The combined organics were dried with anhydroussodium sulfate, filtered and concentrated under reduced pressure. Theresidue was purified by reverse-phase HPLC on a Gilson system (C18column), eluting with 20-80% acetonitrile in water containing 0.1%trifluoroacetic acid, to give the title product.

1.91.113-(1-((3-(3-aminopropyl)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydriosoquinolin-2(1H)-yl)picolinicAcid

To a solution of Example 1.91.9 (350 mg) in dichloromethane (5 mL) wasadded trifluoroacetic acid (5 mL). The mixture was stirred overnight.The mixture was concentrated and the residue was purified by reversephase HPLC using a Gilson system, eluting with 20-80/o acetonitrile inwater containing 0.1% v/v trifluoroacetic acid, to provide the titlecompound. ¹H NMR (500 MHz, DMSO-d₆) δ ppm 12.86 (s, 1H), 8.03 (d, 1H),7.79 (d, 1H), 7.62 (d, 4H), 7.47 (dt, 3H), 7.36 (q, 2H), 7.27 (s, 1H),6.95 (d, 1H), 4.95 (s, 2H), 3.77 (s, 2H), 3.01 (t, 2H), 2.72 (q, 2H),2.09 (s, 3H), 1.45 (t, 2H), 1.18-1.05 (m, 9H), 1.00 (d, 6H), 0.80 (s,6H). MS (ESI) m/e 744.2 (M+H)⁺.

1.91.12 Tert-butyl6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-(3-((2-((4-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylbutoxy)sulfonyl)ethyl)amino)propyl)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinate

The title compound was prepared using the procedure in Example 1.2.8,replacing Example 1.2.7 with Example 1.91.10.

1.91.136-{8-[(1,3-benzothiazol-2-yl)carbamoyl]-3,4-dihydroisoquinolin-2(1H)-yl}-3-{1-[(3,5-dimethyl-7-{3-[(2-sulfoethyl)amino]propyl}tricyclo[3.3.1.1^(3,7)]decan-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid

The title compound was prepared using the procedure in Example 1.2.9,replacing Example 1.2.8 with Example 1.91.12. ¹H NMR (501 MHz, DMSO-d₆)δ 12.85 (s, 1H), 8.02 (dd, 1H), 7.77 (d, 1H), 7.69 (d, 1H), 7.54-7.39(m, 3H), 7.38-7.31 (m 2H), 7.26 (s, 1H), 6.94 (d, 1H), 4.94 (s, 2H),3.87 (t, 2H), 3.15 (p, 2H), 3.00 (t, 2H), 2.86 (dq, 2H), 2.76 (t, 2H),2.08 (s, 3H), 1.47 (td, 2H), 1.08 (d, 9H), 0.99 (d, 7H), 0.79 (s, 7H).MS (ESI) m/e 852.2 (M+H)⁺.

Example 2: Synthesis of Exemplary Synthons

This example provides synthetic methods for exemplary synthons useful tomake ADCs.

2.1 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yL}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide(Synthon CZ)

Example 1.2.9 (100 mg) and4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl) carbonate (purchased from Synchem, 114 mg) inN,N-dimethylformamide (7 mL) was cooled in an water-ice bath, andN,N-diisopropylethylamine (0.15 mL) was added. The mixture was stirredat 0° C. for 30 minutes and then at room temperature overnight. Thereaction was purified by a reverse phase HPLC using a Gilson system,eluting with 20-60% acetonitrile in water containing 0.1% v/vtrifluoroacetic acid, to provide the title compound. ¹H NMR (400 MHz,dimethyl sulfoxide-d₆) δ ppm 12.85 (s, 1H), 9.99 (s, 1H), 8.04 (t, 2H),7.75-7.82 (m, 2H), 7.40-7.63 (m, 6H), 7.32-7.39 (m, 2H), 7.24-7.29 (m.3H), 6.99 (s, 2H), 6.95 (d, 1H), 6.01 (s, 1H), 4.83-5.08 (m, 4H),4.29-4.48 (m, 1H), 4.19 (t, 1H), 3.84-3.94 (m, 2), 3.80 (d, 2H),3.14-3.29 (m, 2H), 2.87-3.06 (m, 4H), 2.57-2.69 (m, 2H), 2.03-2.24 (m,5H), 1.89-2.02 (m, 1H), 1.53-1.78 (m, 2H), 1.26-1.53 (m, 8H), 0.89-1.27(m, 12H), 0.75-0.88 (m. 12H). MS (ESI) m/e 1452.2 (M+H)⁺.

2.2 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](3-sulfopropyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide(Synthon DH)

The title compound was prepared as described in Example 2.1, replacingExample 1.2.9 with Example 1.6.2. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 12.83 (s, 1H), 9.98 (s, 1H), 8.04 (t, 2H), 7.75-7.81(m, 2H), 7.54-7.64 (m, 3H), 7.40-7.54 (m, 3H), 7.32-7.39 (m, 2H),7.24-7.31 (m 3H), 6.93-7.01 (m 3H), 4.86-5.03 (m, 4H), 4.32-4.48 (m,2H), 4.13-4.26 (m, 2H), 3.31-3.45 (m. 4H), 3.24 (d, 4H), 2.88-3.07 (m,4H), 2.30-2.39 (m, 2H), 2.04-2.24 (m, 5H), 1.86-2.03 (m, 1H), 0.89-1.82(m, 27H), 0.74-0.88 (m, 13H). MS (ESI) m/e 1466.3 (M+H)⁺.

2.3 This Paragraph was Intentionally Left Blank 2.4 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-[4-({[{2-[2-((3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)oxy)ethoxy]ethyl}(2-sulfoethyl)carbamoyl]oxy}methyl)phenyl]-N-carbamoyl-L-ornithinamide(Synthon EP)

The title compound was prepared as described in Example 2.1, replacingExample 1.2.9 with Example 1.11.4. ¹H NMR (500 MHz, dimethylsulfoxide-de) ppm 12.85 (s, 1H), 10.00 (s, 1H), 8.01-8.10 (m, 2H), 7.79(dd, 2H), 7.55-7.65 (m, 3H), 7.41-7.53 (m 3H), 7.32-7.38 (m, 2H),7.25-7.30 (m, 3H), 6.97-7.02 (m, 2H), 6.96 (d, 1H), 6.03 (s, 1H),4.90-5.03 (m, 4H), 4.31-4.46 (m, 1H), 4.20 (s, 1H), 3.88 (t, 2H), 3.82(s, 2H), 2.97-3.06 (m, 2H), 2.88-2.98 (m, 1H), 2.58-2.68 (m, 2H),2.05-2.22 (m, 5H), 1.92-2.02 (m, 1H), 0.89-1.75 (m, 23H), 0.77-0.87 (m,12H). MS (ESI) m/e 1496.3 (M+H)⁺.

2.5 Synthesis of methyl6-[4-(3-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]({[4-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N⁵-carbamoyl-L-ornithyl}amino)benzyl]oxy}carbonyl)amino}propyl)-1H-1,2,3-triazol-1-yl]-6-deoxy-beta-L-glucopyranoside(Synthon EF) 2.5.1 pent-4-ynal

To a solution of oxalyl chloride (9.12 mL) dissolved in dichloromethane(200 mL) at −78° C. was added dimethyl sulfoxide (14.8 mL) dissolved indichloromethane (40 mL) over 20 minutes. After the solution was stirredfor an additional 30 minutes. 4-pentynol (8.0 g) dissolved indichloromethane (80 mL) was added over 10 minutes. The reaction mixturewas stirred at −78° C. for an additional 60 minutes. Triethylamine (66.2mL) was added at −78° C., and the reaction mixture was stirred for 60minutes and then allowed to warm to 10° C. over an additional hour.Water (200 mL) was added, and the two layers were separated. The aqueouslayer was acidified with 1% aqueous HCl and then back-extracted withdichloromethane (3× 100 mL). The combined organic layers were washedwith 1% aqueous HCl, and aqueous NaHCO₃. The aqueous extracts wereback-extracted with dichloromethane (2×100 mL), and the combined organicextracts were washed with brine and dried over sodium sulfate. Afterfiltration, the solvent was removed by rotary evaporation (30° C. waterbath) to provide the title compound.

2.5.26-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3,5-dimethyl-7-(2-(pent-4-yn-1-ylamino)ethoxy)adamantan-1-yl)methyl)-5-methyl-11H-pyrazol-4-yl)picolinicAcid

To a solution of Example 1.2.7 (85 mg) in tetrahydrofuran (2 mL) wasadded pent-4-yanl (8.7 mg), acetic acid (20 mg) and sodium sulfate (300mg). The mixture was stirred for 1 hour, and sodiumtriacetoxyborohydride (45 mg) was added to the reaction mixture. Themixture was stirred overnight, then diluted with ethyl acetate (200 mL),washed with water and brine, and dried over sodium sulfate. Filtrationand evaporation of the solvent gave a residue, which was dissolved indimethyl sulfoxide/methanol (1:1.3 mL). The mixture was purified byreverse phase HPLC on a Gilson system, eluting with 10-85% acetonitrilein 0.1% trifluoroacetic acid in water, to give the title compound. MS(ESI) m/e 812.1 (M+H)⁺.

2.5.36-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3,5-dimethyl-7-(2-((3-(1-(((2S,3R,4R,5S,6S)-3,4,5-trihydroxy-6-methoxytetrahydro-2H-pyran-2-yl)methyl)-1H-1,2,3-triazol-4-yl)propyl)amino)ethoxy)adamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinicAcid

To a solution of(2S,3S,4R,5S,6S)-2-(azidomethyl)-6-methoxytetrahydro-2H-pyran-3,4,5-triyltriacetate (8.63 mg) in t-butanol (2 mL) and water (1 mL) was addedExample 2.5.2 (20 mg), copper(II) sulfate pentahydrate (2.0 mg) andsodium ascorbate (5 mg). The mixture was stirred 20 minutes at 100° C.under microwave conditions (Biotage Initiator). Lithium hydroxidemonohydrate (50 mg) was added to the mixture, and it was stirredovernight. The mixture was neutralized with trifluoroacetic acid andpurified by reverse phase HPLC (Gilson system), eluting with 10-85%acetonitrile in 0.1% trifluoroacetic acid in water, to provide the titlecompound. MS (ESI) m/e 1032.2 (M+H)⁺.

2.5.4 methyl6-[4-(3-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]({[4-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valy-N⁵-carbamoyl-L-ornithyl}amino)benzyl]oxy}carbonyl)amino}propyl)-1H-1,2,3-triazol-1-yl]-6-deoxy-beta-L-glucopyranoside

To a solution of4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl4-nitrophenyl carbonate (7.16 mg) and Example 2.5.3 (10 mg) inN,N-dimethylformamide (2 mL) was added N,N-diisopropylethylamine (0.1mL). The mixture was stirred overnight, then acidified withtrifluoroacetic acid and purified by reverse phase HPLC (Gilson system),eluting with 10-85% acetonitrile in 0.1% trifluoroacetic acid in water,to provide the title compound. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δppm 9.65 (s, 1H), 7.97 (d, 1H), 7.76 (d, 1H), 7.64-7.72 (m, 2H),7.53-7.63 (m, 3H), 7.38-7.51 (m, 4H), 7.30-7.37 (m, 2H), 7.22-7.27 (m,3H), 6.84-6.98 (m, 3H), 4.97 (d, 4H), 4.65 (dd, 1H), 4.50 (d, 1H),4.36-4.46 (m, 1H), 4.25-4.32 (m, 1H), 4.10-4.20 (m, 1H), 3.85-3.95 (m2H), 3.79 (s, 2H), 3.66-3.73 (m, 2H), 2.99-3.03 (m, 7H), 2.57 (t, 3H),2.12-2.22 (m, 3H), 2.08 (s, 3H), 1.99-2.05 (m, 2H), 1.70-1.88 (m, 4H),1.39-1.67 (m, 8H), 1.35 (s, 3H), 0.92-1.28 (m, 14H), 0.80-0.88 (m, 16H).MS (ESI) m/e 1629.5 (M+H)+.

2.6 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-(4-{[([2-({3-[(4-{(6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]{3-[1-(beta-D-glucopyranuronosyl)-1H-1,2,3-triazol-4-yl]propyl}carbamoyl)oxy]methyl}phenyl)-N⁵-carbamoyl-L-ornithinamide (Synthon EG) 2.6.16-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-(2-((3-(1-((2R,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)-1H-1,2,3-triazol-4-yl)propyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinicAcid

To a solution of(2R,3R,4S,5S,6S)-2-azido-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (8.63 mg) in t-butanol (2 mL) and water (1 mL) was addedExample 2.5.2 (20 mg), copper(II) sulfate pentahydrate (2.0 mg) andsodium ascorbate (5 mg). The mixture was stirred 20 minutes at 100° C.under microwave conditions (Biotage Initiator). Lithium hydroxidemonohydrate (50 mg) was added to the mixture, and it was stirredovernight. The mixture was neutralized with trifluoroacetic acid andpurified by reverse phase HPLC (Gilson system) eluting with 10-85%acetonitrile in 0.1% trifluoroacetic acid in water, to provide the titlecompound. MS (ESI) m/e 1032.1 (M+H)⁺.

2.6.2N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-(4-{[([2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]{3-[1-(beta-D-glucopyranuronosyl)-1H-1,2,3-triazol-4-yl]propyl}carbamoyl)oxy]methyl}phenyl)-N⁵-carbamoyl-L-ornithinamide

The title compound was prepared by substituting Example 2.6.1 forExample 2.5.3 in Example 2.5.4. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆)δ ppm 9.64 (s, 1H), 7.98 (d, 1), 7.90 (s, 1H), 7.76 (d, 1H), 7.68 (s.1H), 7.52-7.62 (m, 3H), 7.20-7.50 (m, 9H), 6.84-6.98 (m, 3H), 5.56 (d,1H), 4.98 (d, 4H), 4.36-4.49 (m, 2H), 4.11-4.23 (m, 2H), 3.96 (d, 2H),3.74-3.91 (m, 7H), 3.51-3.58 (m, 5H), 3.35-3.49 (m, 10H), 2.97-3.02 (m,6H), 2.57-2.66 (m, 3H), 2.12-2.24 (m, 2H), 2.08 (s, 3H), 1.69-2.01 (m,3H), 1.35-1.65 (m, 9H), 0.93-1.28 (m, 10H), 0.81-0.89 (m, 10H). MS (ESI)m/e 1629.4 (M+H)⁺.

2.7 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[(2R)-1-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](methyl)amino}-1-oxo-3-sulfopropan-2-yl]carbamoyl}oxy)methyl]phenyl}-L-alaninamide(Synthon EH)

To a solution of Example 1.13.8 (0.018 g) and4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)benzyl(4-nitrophenyl) carbonate (0.015 g, 0.023 mmol) in N,N-dimethylformamide(0.75 mL) was added N,N-diisopropylethylamine (0.015 mL). After stirringovernight, the reaction was diluted with N,N-dimethylformamide (0.75 mL)and water (0.5 mL). The mixture was purified by reverse phase HPLC usinga Gilson system, eluting with 10-70% acetonitrile in water containing0.1% v/v trifluoroacetic acid. The desired fractions were combined andfreeze-dried to provide the title compound. ¹H NMR (500 MHz, dimethylsulfoxide-d₆) δ ppm 12.86 (s, 1H), 9.93 (s, 1H), 8.14 (d, 1H), 8.04 (d,1H), 7.84-7.76 (m, 2H), 7.61 (d, 1H), 7.57 (d, 2H), 7.53 (dd, 1H), 7.47(t, 1H), 7.43 (d, 1H), 7.39-7.30 (m, 4H), 7.26 (d, 2H), 6.99 (s, 2H),6.97 (dd, 1H), 4.96 (s, 2H), 4.90 (t, 2H), 4.75-4.65 (m, 1H), 4.46-4.33(m, 2H), 4.17 (dd, 2H), 3.66-3.47 (m, 4H), 3.36 (t, 4H), 3.12 (s, 2H),3.01 (t, 2H), 2.85-2.60 (m, 4H), 2.25-2.05 (m, 5H), 2.05-1.90 (m, 1H),1.58-0.76 (m, 32H). MS (ESI) m/e 1423.2 (M+H)⁺.

2.8 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl][4-(beta-D-glucopyranosyloxy)benzyl]carbamoyl}oxy)methyl]phenyl}-N-carbamoyl-L-ornithinamide(Synthon ER) 2.8.16-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3,5-dimethyl-7-(2-((4-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)amino)ethoxy)adamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinicAcid

To a solution of Example 1.2.7 (44.5 mg) in tetrahydrofuran (2 mL) andacetic acid (0.2 mL) was added4-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)benzaldehyde(17 mg) and MgSO₄ (300 mg). The mixture was stirred for 1 hour beforethe addition of sodium cyanoborohydride on resin (300 mg). The mixturewas stirred overnight. The mixture was filtered, and the solvent wasevaporated. The residue was dissolved in dimethyl sulfoxide/methanol(1:1, 4 mL) and purified by reverse phase HPLC (Gilson system), elutingwith 10-85% acetonitrile in 0.1% trifluoroacetic acid in water, to givethe title compound. MS (ESI) m/e 1015.2 (M+H)⁺.

2.8.2N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1,]dec-1-yl}oxy)ethyl][4-(beta-D-glucopyranosyloxy)benzyl]carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide

The title compound was prepared by substituting Example 2.8.1 forExample 2.5.3 in Example 2.5.4. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆)δ ppm 12.87 (s, 1H), 10.00 (s, 1H), 7.96-8.14 (m, 2H), 7.79 (d, 2H),7.55-7.68 (m, 3H), 7.09-7.52 (m, 1H), 6.91-7.01 (m, 5H), 5.09 (d, 1H),4.95 (dd, 4H), 4.35-4.47 (m, 4H), 4.14-4.23 (m, 3H), 3.86-3.94 (m, 6H),3.31-3.46 (m, 8H), 3.16-3.25 (m, 3H), 2.90-3.04 (m, 4H), 2.59 (s, 1H),1.88-2.24 (m, 6H), 0.88-1.75 (m, 24H), 0.76-0.90 (m, 12H). MS (ESI) m/e1613.7 (M+H)⁺.

2.9 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[4-(beta-D-allopyranosyloxy)benzyl][(2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide(Synthon ES) 2.9.16-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3,5-dimethyl-7-(2-((4-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)amino)ethoxy)adamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinicAcid

To a solution of Example 1.2.7 (44.5 mg) in tetrahydrofuran (2 mL) andacetic acid (0.2 mL) was added4-(((2S,3R,4R5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)benzaldehyde(17 mg) and MgSO₄ (300 mg). The mixture was stirred for 1 hour beforethe addition of sodium cyanoborohydride on resin (300 mg). The mixturewas stirred overnight. The mixture was filtered, and the solvent wasevaporated. The residue was dissolved in dimethyl sulfoxide/methanol(1:1, 4 mL) and purified by reverse phase HPLC (Gilson system), elutingwith 10-85% acetonitrile in 0.1% trifluoroacetic acid in water, to givethe title compound. MS (ESI) m/e 1015.2 (M+H)⁺.

2.9.2N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[4-(beta-D-allopyranosyloxy)benzyl][2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide

The title compound was prepared by substituting Example 2.9.1 forExample 2.5.3 in Example 2.5.4. ¹H NMR (500 MHz, dimethyl sulfoxide-d₆)δ ppm 12.86 (s, 1H), 10.00 (s, 1H), 7.96-8.11 (m, 2H), 7.79 (d, 2H),7.53-7.65 (m, 3H), 7.08-7.52 (m, 10H), 6.91-7.00 (m, 5H), 5.09 (d, 1H),4.99 (d, 4H), 4.35-4.48 (m, 3H), 4.13-4.23 (m, 2f). 3.82-3.96 (m, 8H),3.32-3.50 (m, 10H), 3.12-3.25 (m, 3H), 2.90-3.06 (m, 5H), 1.89-2.19 (m,6H), 0.88-1.75 (m, 22H), 0.76-0.88 (m, 1H). MS (ESI) m/e 1612.5 (M+H)⁺.

2.10 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)-]dec-1-yl}oxy)ethyl](2-phosphonoethyl)carbamoyl}oxy)methyl]phenyl}-N-carbamoyl-L-ornithinamide(Synthon EQ)

The title compound was prepared as described in Example 2.1, replacingExample 1.2.9 with Example 1.12.2. ¹H NMR (500 MHz, dimethylsulfoxide-d₆) δ ppm 9.99 (s, 1H), 8.01-8.09 (m, 2H), 7.76-7.81 (m, 2H),7.56-7.64 (m, 3H), 7.41-7.53 (m, 3H), 7.36 (q, 2H), 7.25-7.30 (m, 3H),6.99 (s, 2H), 6.94 (d, 1H), 5.98 (s, 1H), 4.89-5.07 (m, 4H), 4.38 (s,1H), 4.19 (t, 1H), 3.88 (t, 2H), 3.80 (d, 2H), 2.89-3.08 (m, 5H),2.04-2.24 (m, 5H), 1.89-2.02 (m, 1H), 1.76-1.87 (m, 2H), 0.89-1.72 (m,23H), 0.78-0.88 (m, 12H). MS (ESI) m/e 1452.2 (M+H)⁺.

2.11 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydriosoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-phosphonoethyl)carbamoyl}oxy)methyl]phenyl}-L-alaninamide(Synthon EU)

The title compound was prepared as described in Example 2.1, replacingExample 1.2.9 and4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl) carbonate with Example 1.12.2 and4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)benzyl(4-nitrophenyl) carbonate, respectively. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 9.93 (s, 1H), 8.12 (d, 1H), 8.03 (d, 1H), 7.72-7.83(m, 2H), 7.54-7.65 (m, 3H), 7.41-7.54 (m, 3H), 7.31-7.40 (m, 2H),7.24-7.30 (m, 3H), 6.99 (s, 2H), 6.94 (d, 1H), 4.87-5.11 (m, 3H),4.11-4.45 (m, 1H), 3.88 (t 2H), 3.79 (d, 2H), 2.97-3.05 (m, 2H),2.63-2.70 (m, 1H), 2.29-2.37 (m, 1H), 2.03-2.20 (m, 5H), 1.73-2.00 (m,5H), 1.39-1.55 (m, 4H), 0.88-1.38 (m, 19H), 0.72-0.89 (m, 12H). MS (ESI)m/e 1364.5 (M−H)⁻.

2.12 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](3-phosphonopropyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide(Synthon EV)

The title compound was prepared as described in Example 2.1, replacingExample 1.2.9 with Example 1.14.4. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 9.98 (s, 1H), 8.04 (t 2H), 7.78 (t, 2H), 7.61 (t,3H), 7.39-7.54 (m, 3H), 7.32-7.39 (m, 2H), 7.25-7.30 (m, 3H), 6.99 (s,2H), 6.95 (d, 1H), 6.01 (s, 1H), 4.97 (d, 4H), 4.29-4.47 (m, 2H),4.14-4.23 (m, 2H), 3.85-3.93 (m, 2H), 3.32-3.42 (m, 2H), 3.24 (s, 2H),2.88-3.09 (m, 3H), 1.87-2.23 (m, 6H), 0.91-1.74 (m, 27H), 0.72-0.89 (m,12H). MS (ESI) m/e 1466.3 (M+H)⁺.

2.13 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[(2R)-1-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]amino}-1-oxo-3-sulfopropan-2-yl]carbamoyl}oxy)methyl]phenyl}-L-alaninamide(Synthon EW)

To a solution of Example 1.15 (0.020 g) and4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)benzyl(4-nitrophenyl) carbonate (0.017 g) in N,N-dimethylformamide (0.5 mL)was added N,N-diisopropylethylamine (0.017 mL). The reaction was stirredovernight and was diluted with N,N-dimethylformamide (1 mL), water (0.5mL). The mixture was purified by reverse phase HPLC using a Gilsonsystem, eluting with 10-70% acetonitrile in water containing 0.1% v/vtrifluoroacetic acid. The desired fractions were combined andfreeze-dried to provide the title compound. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 12.85 (s, 1H), 9.93 (s, 1H), 8.12 (d, 1H), 8.04 (d,1H), 7.86-7.76 (m, 3H), 7.63-7.41 (m, 7H), 7.39-7.32 (m, 2H), 7.30 (s,1H), 7.30-7.21 (m, 2H), 6.99 (s, 2H), 6.97 (d, 1H), 4.96 (s, 2H), 4.93(s, 2H), 4.49-4.33 (m, 2H), 4.18 (dd, 2H), 4.15-4.08 (m, 2H), 3.90-3.86(m, 2H), 3.36 (t, 2H), 3.34-3.27 (m, 1H), 3.18-3.04 (m, 2H), 3.04-2.96(m 2H), 2.89-2.61 (m, 2H), 2.27-2.05 (m, 5H), 2.03-1.87 (m, 1H),1.59-1.42 (m, 4H), 1.42-0.91 (m 18H), 0.91-0.76 (m, I1H). MS (−ESI) m/e1407.5 (M−H)⁻.

2.14 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-[4-({[{2-[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethoxy]ethyl}(3-phosphonopropyl)carbamoyl]oxy}methyl)phenyl]-N⁵-carbamoyl-L-ornithinamide(Synthon EX)

A mixture of Example 1.16.2 (59 mg),4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl) carbonate (48 mg), and N,N-diisopropylethylamine (0.056mL) in 2 mL N,N-dimethylformamide was stirred for 24 hours. The mixturewas purified via reverse phase chromatography on a Biotage Isolera Onesystem using a 40 g C18 column, eluting with 10-90% acetonitrile in 0.1%trifluoroacetic acid/water. The desired fractions were concentrated andthe product was lyophilized from water and 1,4-dioxane to give the titlecompound as a trifluoroacetic acid salt. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 9.97 (bs, 1H), 8.04 (m, 2H), 7.79 (d, 2H), 7.59 (m,3H), 7.46 (m, 3H), 7.36 (m, 2H), 7.27 (m 2H), 6.99 (s, 2H), 6.94 (d,1H), 4.97 (m 4H), 4.40 (m, 2H), 4.17 (dd, 2H), 3.50-4.10 (m, 6H), 3.45(m, 2H), 3.40 (m, 2H), 3.26 (m, 2H), 3.01 (m, 2H), 2.95 (s, 2H), 2.79(s, 2H), 2.15 (m, 2H), 2.09 (s, 2H), 1.68 (m, 2H), 1.60 (m, 1-2H),1.35-1.50 (m, 6H), 1.25 (m, 4H), 1.17 (m, 2H), 1.10 (m, 2H), 0.97 (m,1-2H), 0.84 (m, 12H). MS (ESI) m/e 1510.4 (M+H)⁺.

2.15 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-y)hexanyl]-L-valyl-N-[4-({[{2-[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethoxy]ethyl}(3-phosphonopropyl)carbamoyl]oxy}methyl)phenyl]-L-alaninamide (Synthon EY)

A mixture of Example 1.16.2 (59 mg).4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)benzyl(4-nitrophenyl) carbonate (42 mg), and N,N-diisopropylethylamine (0.042mg) in 2 mL N,N-dimethylformamide was stirred for 24 hours. The mixturewas purified via reverse phase chromatography on a Biotage Isolera Onesystem using a 40 g C18 column, eluting with 10-90% acetonitrile in 0.1%trifluoroacetic acid/water. Fractions were concentrated and the productwas lyophilized from water and 1,4-dioxane to give the title compound asa trifluoroacetic acid salt. MS (ESI) m/e 1422.6 (M−H)⁺.

2.16 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](3-phosphonopropyl)carbamoyl}oxy)methyl]phenyl}-L-alaninamide(Synthon EZ)

A mixture of Example 1.14.4 (50 mg),4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)benzyl(4-nitrophenyl) carbonate (38 mg), and N,N-diisopropylethylamine (0.050mL) in 2 mL N,N-dimethylformamide was stirred for 24 hours. The mixturewas purified via reverse phase chromatography on a Biotage Isolera Onesystem using a 40 g C18 column, eluting with 10-90% acetonitrile in 0.1%trifluoroacetic acid/water. The desired fractions were concentrated andthe product was lyophilized from water and 1,4-dioxane to give the titlecompound as a trifluoroacetic acid salt. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ 6 ppm 9.94 (bs, 1H), 8.12 (d, 1H), 8.04 (d, 1H), 7.80(d, 2H), 7.61 (m, 3H), 7.47 (m, 3H), 7.36 (m, 2H), 7.29 (m, 2H), 6.99(s, 2H), 6.95 (d, 1H), 4.97 (m, 4H), 4.40 (m, 2H), 4.16 (dd, 2H),3.50-4.10 (m, 6H), 3.68 (m, 2H), 3.55 (m, 2H), 3.25 (m, 4H), 3.02 (m,2H), 2.94 (s, 2H), 2.79 (s, 2H), 2.15 (m, 1H), 2.08 (s, 2H), 1.65 (m,2H), 1.40-1.50 (m, 6H), 1.20-1.30 (m, 6H), 1.08-1.19 (m, 4H), 0.97 (m,1-2H), 0.76-0.89 (m, 12H). MS (ESI) m/e 1380.3 (M+H)⁺.

2.17 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3-(2-{[(2S)-3-carboxy-2-({[(4-{[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl)oxy]carbonyl}amino)propanoyl](methyl)amino}ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylicAcid (Synthon FD)

To a solution of Example 1.17 (0.040 g) and4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)benzyl(4-nitrophenyl) carbonate (0.034 g) in N,N-dimethylformamide (1 mL) wasadded N,N-diisopropylethylamine (0.035 mL). The reaction was stirredovernight and diluted with N,N-dimethylformamide (1 mL) and water (0.5mL). The mixture was purified by reverse phase HPLC using a Gilsonsystem, eluting with 10-70% acetonitrile in water containing 0.1% v/vtrifluoroacetic acid. The desired fractions were combined andfreeze-dried to provide the title compound. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 12.84 (s, 1H), 9.92 (s, 1H), 8.13 (d, 1H), 8.03 (d,1H), 7.79 (d, 2H), 7.62 (d, 1H), 7.57 (d, 2H), 7.54-7.41 (m, 3H),7.40-7.32 (m, 2H), 7.31-7.23 (m, 4H), 6.99 (s, 2H), 6.95 (dd, 1H),5.01-4.89 (m, 4H), 4.78 (dq, 1H), 4.45-4.30 (m, 1H), 4.23-4.11 (m, 1H),3.88 (t, 2H), 3.80 (s, 2H), 3.42-3.26 (m, 6H), 3.06 (s, 1H), 3.01 (t,2H), 2.80 (s, 2H), 2.76-2.62 (m, 1H), 2.46-2.36 (m, 1H), 2.25-2.05 (m,5H), 2.05-1.92 (m, 1H), 1.58-1.42 (m, 4H), 1.42-0.91 (m, 20H), 0.91-0.78(m, 9H). MS (ESI) m/e 1387.4 (M+H)⁺.

2.18 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl][4-(beta-D-glucopyranuronosyloxy)benzyl]carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide(Synthon FS)

The title compound was prepared by substituting Example 1.19.2 forExample 2.5.3 in Example 2.5.4. ¹H NMR (500 MHz, dimethyl sulfoxide-d₆)δ ppm 12.86 (s, 1H), 10.00 (s, 1H), 7.97-8.14 (m, 2H), 7.79 (d, 2H),7.07-7.65 (m, 13H), 6.87-7.01 (m, 4H), 5.92-6.08 (m, 1H), 4.87-5.07 (m,4H), 4.33-4.48 (m, 3H), 4.13-4.26 (m, 1H), 3.74-3.94 (m, 6H), 3.14-3.34(m, 8H), 2.84-3.05 (m, 6H), 1.87-2.25 (m, 6H), 0.89-1.73 (m, 21H),0.76-0.87 (m, 12H). MS (ESI) m/e 1626.4 (M+H)⁺.

2.19 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{(6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-phosphonoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide(Synthon F)

The title compound was prepared as described in Example 2.1, replacingExample 1.2.9 with Example 1.20.11. ¹H NMR (500 MHz, dimethylsulfoxide-d₆) δ ppm 10.00 (s, 1H), 8.40 (s, 1H), 8.07 (d, 1H), 8.00 (d,1H), 7.84-7.90 (m, 1H), 7.79 (dd, 3H), 7.55-7.66 (m, 2H), 7.46 (s, 2H),7.37 (t, 1H), 7.29 (t, 3H), 7.18-7.25 (m, 1H), 6.99 (s, 2H), 5.99 (s,1H), 5.00 (d, 1H), 4.38 (s, 1H), 4.13-4.24 (m, 1H), 3.96 (s, 2H), 3.87(d, 2H), 2.88-3.08 (m, 4H), 2.84 (q, 2H), 2.04-2.26 (m, 5H), 1.89-2.01(m, 3H), 1.75-1.88 (m, 2H), 1.63-1.74 (m, 1H), 0.91-1.63 (m, 21f),0.76-0.89 (m, 12H). MS (ESI) m/e 1450.5 (M−H)⁻.

2.20 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N⁵-carbamoyl-N-{4-[({[2-(13-[(4-{2-carboxy-6-[8-([1,3]thiazolo[5,4-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridin-3-yl)-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-L-ornithinamide(Synthon FV)

The title compound was prepared by substituting Example 1.22.5 forExample 1.2.9 in Example 2.1. ¹H NMR (500 MHz, dimethyl sulfoxide-d₆) δppm 13.00 (v br s, 1H), 10.00 (s, 1H), 8.52 (dd, 1H), 8.16 (dd, 1H),8.06 (d, 1H), 7.78 (d, 1H), 7.62 (d, 1H), 7.59 (br m, 2H), 7.53 (m, 2H),7.45 (d, 1H), 7.37 (t, 1H), 7.30 (s, 1H) 7.27 (d, 2H), 6.99 (s, 2H),6.97 (d, 1H), 4.98 (m, 4H), 4.39 (m, 1H), 4.19 (br m, 1H), 3.88 (t, 2H),3.80 (br d, 2H), 3.44, 3.36 (br m, m, total 6H), 3.24 (m, 2H), 2.94-3.01(m, 4H), 2.63 (br m, 2H), 2.14 (m, 2H), 2.10 (s, 3H), 1.97 (br m, 1H),1.68 (br m, 1H), 1.58 (br m. 1H), 1.34-1.47 (m, 8H), 1.08-1.23 (m 10H),0.95 (br m, 2H), 0.85-0.80 (m, 12H). MS (ESI) m/e 1451.4 (M−H)⁻.

2.21 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[(2R)-1-{[2-({3-[(4-{6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](methyl)amino}-1-oxo-3-sulfopropan-2-yl]carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide(Synthon GC)

The title compound was prepared as described in Example 2.1, replacingExample 1.2.9 with Example 1.21.7. ¹H NMR (500 MHz, dimethylsulfoxide-d₆) δ ppm 9.98 (s, 1H), 8.40 (s, 1H), 8.07 (d, 1H), 8.01 (dd,1H), 7.89 (t, 1H), 7.74-7.84 (m, 3H), 7.58 (d, 2H), 7.47 (s, 2H), 7.37(t, 1H), 7.19-7.33 (m, 5H), 7.00 (s, 2H), 4.91 (q, 2H), 4.64-4.76 (m,2H), 4.33-4.43 (m, 2H), 4.15-4.24 (m. 2H), 3.92-4.03 (m, 2H), 3.88 (s,2H), 3.32-3.50 (m, 6H), 3.10-3.22 (m, 2H), 2.89-3.07 (m, 2H), 2.70-2.89(m, 4H), 2.60-2.70 (m, 1H), 2.05-2.28 (m, 5H), 1.90-2.03 (m, 3H),1.64-1.77 (m, 1H), 1.53-1.65 (m, 1H), 0.92-1.53 (m, 21H), 0.77-0.92 (m,12H). MS (ESI) m/e 1507.3 (M−H)⁻.

2.22 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[(2R)-1-{[2-({3-[(4-{6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](methyl)amino}-1-oxo-3-sulfopropan-2-yl]carbamoyl}oxy)methyl]phenyl}-L-alaninamide(Synthon GB)

The title compound was prepared as described in Example 2.1, replacingExample 1.2.9 and4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl) carbonate with Example 1.21.7 and4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)benzyl(4-nitrophenyl) carbonate, respectively. ¹H NMR (500 MHz, dimethylsulfoxide-d₆) δ ppm 9.93 (s, 1H), 8.39 (s, 1H), 8.13 (d, 1H), 8.01 (dd,1H), 7.88 (t, 1H), 7.74-7.84 (m, 3H), 7.57 (d, 2H), 7.46 (s, 2H), 7.37(t, 1H), 7.17-7.33 (m, 5H), 6.99 (s, 2H), 4.91 (d, 2H), 4.65-4.76 (m,1H), 4.30-4.51 (m, 1H), 4.13-4.21 (m, 1H), 3.92-4.00 (m, 2H), 3.88 (s,2H), 3.29-3.46 (m, 4H), 2.93-3.21 (m, 3H), 2.68-2.88 (m, 4H), 2.58-2.68(m, 1H), 2.04-2.26 (m, 5H), 1.89-2.02 (m, 3H), 1.37-1.54 (m, 6H),0.92-1.34 (m, 15H), 0.75-0.91 (m, 12H). MS (ESI) m/e (M+H)⁺.

2.23 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-carbamoyl-N-{4-{([2-({3-[(4-{[2-carboxy-6-[8-([1,3]thiazolo[4,5-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-L-ornithinamide(Synthon FW)

The title compound was prepared by substituting Example 1.23.4 forExample 1.2.9 in Example 2.1. ¹H NMR (500 MHz, dimethyl sulfoxide-d₆) δppm 13.38 (v br s, 1H), 10.00 (s, 1H), 8.66 (m, 2H), 8.06 (d, 1H), 7.78(d, 1H), 7.65 (d, 1H), 7.59 (br m, 2H), 7.53 (m, 1H), 7.47 (m 2H), 7.37(t, 1H), 7.30 (s, 1H) 7.27 (d, 2H), 6.99 (s, 2H), 6.97 (d, 1H), 4.98 (m,4H), 4.39 (m, 1H), 4.19 (br m, 1H), 3.88 (t, 2H), 3.80 (br d, 2H), 3.40(br m, 6H), 3.24 (m, 2H), 2.98 (m, 4H), 2.63 (m, 2H), 2.16 (m, 2H), 2.10(s, 3H), 1.97 (br m, 1H), 1.68 (br m, 1H), 1.58 (br m, H), 1.34-1.47 (m,8H), 1.08-1.23 (m, 10H), 0.95 (br m, 2H), 0.85-0.80 (m, 12H). MS (ESI)m/e 1451.5 (M−H)⁻.

2.24 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide(Synthon GD)

The title compound was prepared as described in Example 2.1, replacingExample 1.2.9 with Example 1.24.2. ¹H NMR (500 MHz, dimethylsulfoxide-d₆) δ ppm 10.00 (s, 1H), 8.38 (s, 1H), 8.07 (d, 1H), 8.00 (d,1H), 7.85-7.92 (m, 1H), 7.73-7.85 (m, 3H), 7.55-7.65 (m, 2H), 7.46 (s,2H), 7.37 (t, 1H), 7.28 (t, 3H), 7.22 (t, 1H), 6.99 (s, 2H), 6.00 (s,1H), 4.99 (d, 1H), 4.28-4.50 (m, 1H), 4.19 (s, 1H), 3.77-4.03 (m, 4H),3.31-3.41 (m, 2H), 3.20-3.29 (m, 2H), 2.87-3.08 (m, 3H), 2.83 (t, 2H),2.63 (d, 2H), 2.05-2.25 (m, 5H), 1.88-2.01 (m, 3H), 1.69 (t, 1H),1.53-1.63 (m, 1H), 1.31-1.53 (m, 8H), 1.04-1.29 (m, 11H), 0.89-1.02 (m,2H), 0.77-0.88 (m, 12H). MS (ESI) m/e 1450.4 (M−H)⁻.

2.25 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-carboxyethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide(Synthon GK)

The title compound was prepared as described in Example 2.1, replacingExample 1.2.9 with Example 1.25.2. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 12.85 (s, 1H), 9.98 (s, 1H), 8.04 (t, 2H), 7.75-7.82(m, 2H), 7.60 (t, 3H), 7.41-7.53 (m, 3H), 7.32-7.39 (m, 2H), 7.24-7.29(m, 3H), 6.99 (s, 2H), 6.94 (d, 3H), 5.97 (s, 1H), 4.88-5.04 (m, 4H),4.38 (d, 1H), 4.12-4.24 (m, 1H), 3.88 (t, 2H), 3.75-3.84 (m, 2H),3.32-3.40 (m, 2H), 3.28 (d, 2H), 2.90-3.05 (m, 4H), 2.42-2.49 (m, 2H),2.05-2.22 (m, 5H), 1.87-2.01 (m, 1H), 0.90-1.76 (m, 22H), 0.74-0.88 (m,12H). MS (ESI) m/e 1414.5 (M−H)⁻.

2.26 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-carboxyethyl)carbamoyl}oxy)methyl]phenyl}-L-alaninamide(Synthon GJ)

The title compound was prepared as described in Example 2.1, replacingExample 1.2.9 and4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl) carbonate with Example 1.25.2 and4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)benzyl(4-nitrophenyl) carbonate, respectively. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 12.78 (s, 1H), 9.93 (s, 1H), 8.12 (d, 1H), 8.03 (d,1H), 7.75-7.83 (m, 2H), 7.54-7.65 (m, 3H), 7.41-7.52 (m, 3H), 7.32-7.40(m, 2H), 7.24-7.29 (m, 3H), 6.98 (s, 2H), 6.94 (d, 1H), 4.90-5.04 (m,4H), 4.32-4.45 (m, 2H), 4.12-4.21 (m, 2H), 3.88 (t, 2H), 3.79 (d, 2H),3.31-3.46 (m, 4H), 3.23-3.31 (m, 2H), 3.01 (t, 2H), 2.46 (t, 2H),2.04-2.22 (m, 5H), 1.87-2.02 (m, 1H), 1.40-1.60 (m, 4H), 0.91-1.37 (m,17H), 0.76-0.88 (m, 12H). MS (ESI) m/e 1328.4 (M−H)⁻.

2.27 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3-(2-{[(2R)-3-carboxy-2-({[(4-{[(2S)-2-([(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino)-3-methylbutanoyl]amino}propanoyl]amino}benzyl)oxy]carbonyl}amino)propanoyl](methyl)amino}ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylicAcid (Synthon GW)

A solution of Example 1.27 (0.043 g) in N,N-dimethylformamide (0.5 mL)was added4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)benzyl(4-nitrophenyl) carbonate (0.042 g) followed byN,N-diisopropylethylamine (0.038 mL), and the reaction was stirred atroom temperature. After stirring for 16 hours, the reaction was dilutedwith water (0.5 mL) and N,N-dimethylformamide (1 mL). The mixture waspurified by reverse phase HPLC using a Gilson system, eluting with10-70% acetonitrile in water containing 0.1% v/v trifluoroacetic acid.The desired fractions were combined and freeze-dried to provide thetitle compound. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ δ ppm 13.05(s, 1H), 10.15 (s, 1H), 8.36 (d, 1H), 8.26 (d, 1H), 8.02 (d, 2H),7.95-7.77 (m, 4H), 7.77-7.63 (m, 3H), 7.63-7.54 (m, 2H), 7.54-7.46 (m,3H), 7.22 (s, 2H), 7.18 (dd, 1H), 5.17 (d, 4H), 5.01 (dq, 1H), 4.61 (p,1H), 4.39 (t, 1H), 4.11 (t, 2H), 4.03 (s, 2H), 3.64-3.49 (m, 2H), 3.29(s, 1H), 3.24 (t, 2H), 3.03 (s, 2H), 2.92 (dt. 1H), 2.73-2.61 (m, 4H),2.35 (d, 4H), 2.18 (dt, 1H), 1.71 (h, 4H), 1.65-1.13 (m, 18H), 1.13-1.01(m, 13H). MS (ESI) m/e 1387.3 (M+H)⁺.

2.28 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl][1-(carboxymethyl)piperidin-4-yl]carbamoyl}oxy)methyl]phenyl}-N-carbamoyl-L-ornithinamide(Synthon HF)

A solution of Example 1.28 (0.0449 g),4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl) carbonate (0.049 g) and N,N-diisopropylethylamine (0.044mL) were stirred together in N,N-dimethylformamide (0.5 mL) at roomtemperature. The reaction mixture was stirred overnight and diluted withN,N-dimethylformamide (1 mL) and water (0.5 mL). The mixture waspurified by reverse phase HPLC using a Gilson system, eluting with10-90% acetonitrile in water containing 0.1% v/v trifluoroacetic acid.The desired fractions were combined and freeze-dried to provide thetitle compound. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm 12.85 (s,1H), 9.99 (s, 1H), 8.04 (t, 2H), 7.78 (t, 2H), 7.65-7.58 (m, 3H),7.54-7.41 (m, 3H), 7.38 (d, 1H), 7.34 (d, 1H), 7.32-7.24 (m. 3H), 6.99(s, 2H), 6.95 (d, 1H), 5.97 (s, 1H), 5.01 (s, 2H), 4.96 (s, 2H), 4.38(q, 1H), 4.23-4.14 (m, 1H), 4.05 (s, 2H), 3.88 (t, 2H), 3.80 (s, 2H),3.36 (t, 2H), 3.26-2.86 (m, 8H), 2.27-2.02 (m, 6H), 2.02-1.86 (m, 2H),1.86-1.75 (m, 2H), 1.75-1.54 (m, 2H), 1.54-0.90 (m, 24H), 0.89-0.72 (m,14H). MS (ESI) m/e 1485.2 (M+H)⁺.

2.29 Synthesis of(S)-6-((2-((3-((4-(6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydriosoquinolin-2(1H)-yl)-2-carboxypyridin-3-yl)-5-methyl-1H-pyrazol-1-yl)methyl)-5,7-dimethyladamantan-1-yl)oxy)ethyl)(methyl)amino)-5-((((4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)amino)-N,N,N-trimethyl-6-oxohexan-1-aminiumSalt (Synthon HG)

A solution of Example 1.29 (8 mg),4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl) carbonate (8.24 mg) and N,N-diisopropylethylamine (7.50μl. 0.043 mmol) in N,N-dimethylformamide (0.250 mL) was stirred at roomtemperature. After 3 hours, the reaction was diluted withN,N-dimethylformamide (1.25 mL) and water (0.5 mL). The mixture waspurified by reverse phase HPLC using a Gilson system, eluting with10-90% acetonitrile in water containing 0.1% v/v trifluoroacetic acid.The desired fractions were combined and freeze-dried to provide thetitle compound. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm 12.85 (s,1H), 9.96 (s, 1H), 8.04 (t, 2H), 7.83-7.76 (m, 2H), 7.66-7.56 (m, 3H),7.53-7.42 (m, 4H), 7.41-7.32 (m, 2H), 7.31-7.23 (m 3H), 6.99 (s, 2H),6.95 (d, 1H), 5.99 (s, 1H), 5.04-4.87 (m, 4H), 4.44-4.33 (m, 2H),4.24-4.12 (m, 2H), 3.88 (t, 2H), 3.81 (s, 2H), 3.50-3.13 (m, 9H),3.11-2.92 (m 14H), 2.80 (s, 1H), 2.25-2.04 (m, 5H), 2.03-1.89 (m, 1H),1.75-0.91 (m, 28H), 0.91-0.77 (m 12H). MS (ESI) m/e 1528.5 (M+H)⁺.

2.30 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-L-alaninamide(Synthon HP)

The title compound was prepared as described in Example 2.1, replacing4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl) carbonate with4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)benzyl(4-nitrophenyl) carbonate. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm12.83 (s, 1H), 9.94 (s, 1H), 8.12 (d, 1H), 8.04 (d, 1H), 7.79 (d, 2H),7.40-7.63 (m, 6H), 7.32-7.39 (m, 2H), 7.24-7.30 (m, 3H), 6.99 (s, 2H),6.95 (d. 1H), 4.90-5.03 (m, 4H), 4.31-4.47 (m, 1H), 4.09-4.24 (m, 1H),3.84-3.93 (m, 2H), 3.81 (s, 2H), 3.30-3.39 (m, 2H), 3.20-3.28 (m, 2H),3.01 (t, 2H), 2.57-2.65 (m, 2H), 2.05-2.22 (m, 5H), 1.87-2.02 (m. 2H),1.41-1.58 (m 4H), 1.22 (d, 18H), 0.74-0.89 (m, 12H). MS (ESI) m/e 1364.5(M−H)⁻.

2.31 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-[4-({[(4-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)-]dec-1-yl}oxy)ethyl](2-sulfoethyl)amino}piperidin-1-yl)carbonyl]oxy}methyl)phenyl]-N⁵-carbamoyl-L-ornithinamide(Synthon HR)

A solution of Example 1.30.2 (0.038 g).4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl) carbonate (0.035 g) and N,N-diisopropylethylamine (0.032mL) in N,N-dimethylformamide (0.5 mL) was stirred at room temperature.After stirring for 3 hours, the reaction was diluted withN,N-dimethylformamide (1.25 mL) and water (0.5 mL). The mixture waspurified by reverse phase HPLC using a Gilson system, eluting with10-90% acetonitrile in water containing 0.1% v/v trifluoroacetic acid.The desired fractions were combined and freeze-dried to provide thetitle compound. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm 9.98 (s,1H), 9.02 (s, 1H), 8.10-8.00 (m, 2H), 7.79 (d, 2H), 7.64-7.56 (m, 3H),7.53 (d, 1H), 7.47 (t, 1H), 7.43 (d, 1H), 7.39-7.32 (m, 2H), 7.29 (d,3H), 6.99 (s. 2H), 6.95 (d, 1H), 6.00 (s, 1H), 4.99 (s, 2H), 4.96 (s,2H), 4.48-4.32 (m, 2H), 4.27-4.15 (m, 2H), 4.11 (d, 2H), 3.88 (t, 2),3.82 (s, 2H), 3.40-3.33 (m, 4H), 3.24-3.11 (m, 2), 3.11-2.72 (m, 8H),2.26-2.04 (m, 4H), 2.04-1.80 (m, 3H), 1.80-0.92 (m, 26H), 0.92-0.77 (m,12H). MS (ESI) m/e 1535.4 (M+H)⁺.

2.32 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-5-(3-phosphonopropoxy)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](methyl)carbamoyl}oxy)methyl]phenyl}-N-carbamoyl-L-ornithinamide(Synthon HU)

The title compound was prepared by substituting Example 1.31.11 forExample 2.5.3 in Example 2.5.4. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆)δ ppm 9.98 (s, 1f). 8.03 (dd, 2H), 7.70-7.84 (m, 3H), 7.59 (d, 2H), 7.48(dd, 2H), 7.23-7.37 (m 4H), 6.93-7.02 (m 4H), 4.99 (d, 4H), 4.12-4.21 (m8H), 3.88-3.96 (m, 4H), 3.75-3.84 (m, 4H), 3.23-3.49 (m, 7H), 2.73-3.07(m 8H), 1.89-2.21 (m, 9H), 0.91-1.77 (m 25H), 0.77-0.91 (m, 12H). MS(ESI) m/e 1496.3 (M+H)⁺.

2.33 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-[4-({[(4-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](3-phosphonopropyl)amino}piperidin-1-yl)carbonyl]oxy}methyl)phenyl]-N-carbamoyl-L-ornithinamide(Synthon HT)

A solution of Example 1.26.2 (0.040 g).4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl) carbonate (0.030 g) and N,N-diisopropylethylamine (0.020mL) in N,N-dimethylformamide (0.5 mL) was stirred at room temperature.After stirring for 3 hours, the reaction was diluted withN,N-dimethylformamide (1.25 mL) and water (0.5 mL). The mixture waspurified by reverse phase HPLC using a Gilson system, eluting with10-90% acetonitrile in water containing 0.1% v/v trifluoroacetic acid.The desired fractions were combined and freeze-dried to provide thetitle compound. ¹H NMR (500 MHz, dimethyl sulfoxide-d₆) δ ppm 9.98 (s,1H), 9.26 (s, 1H), 8.06 (d, 1H), 8.05-8.01 (m, 1H), 7.79 (d, 2H), 7.62(d, 1H), 7.61-7.57 (m, 2H), 7.52-7.42 (m, 3H), 7.38 (d, 1H), 7.35 (d,1H), 7.32-7.26 (m, 3H), 6.99 (s, 2H), 6.95 (d, 1H), 6.01 (s, 1H), 4.99(s, 2H), 4.96 (s, 3H), 4.44-4.33 (m, 2H), 4.18 (dd, 2H), 3.88 (t, 2H),3.83 (s, 2H), 3.71-3.61 (m, 2H), 3.53 (t, 2H), 3.36 (t, 2H), 3.07-2.66(m, 8H), 2.28-2.06 (m, 6H), 2.05-1.92 (m, 2H), 1.92-1.80 (m, 2H),1.78-0.95 (m, 32H), 0.92-0.77 (m, 14H). MS (ESI) m/e 1549.5 (M+H)⁺.

2.34 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](3-phosphonopropyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide(Synthon HV)

The title compound was prepared by substituting Example 1.14.4 forExample 2.5.3 in Example 2.5.4. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆)δ ppm 9.98 (s, 1H), 9.02 (s, 1H), 8.32-8.45 (m, 1H), 8.12-8.27 (m, 3H),7.98-8.09 (m, 3H), 7.93 (d, 1H), 7.66-7.83 (m, 4H), 7.54-7.64 (m, 2H),7.46-7.50 (m, 2H), 7.24-7.40 (m, 3H), 6.99 (s, 2H), 5.93-6.09 (m, 1H),4.99 (s, 3H), 4.33-4.49 (m, 3H), 4.15-4.20 (m, 3H), 3.19-3.50 (m, 10H),2.86-3.07 (m, 3H), 1.87-2.27 (m, 7H), 0.91-1.77 (m, 26H), 0.76-0.89 (m,10H). MS (ESI) m/e 1461.1 (M+H)⁺.

2.35 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-[4-({[(4-{[2-(13-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)-]dec-1-yl}oxy)ethyl](2-carboxyethyl)amino)piperidin-1-yl)carbonyl]oxy}methyl)phenyl]-N⁵-carbamoyl-L-ornithinamide(Synthon HZ)

A solution of Example 1.36.2 (0.031 g),4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl) carbonate (0.025 g) and N,N-diisopropylethylamine (0.016mL) in N,N-dimethylformamide (0.5 mL) was stirred at room temperature.After stirring for 3 hours, the reaction was diluted withN,N-dimethylformamide (1.25 mL) and water (0.5 mL). The mixture waspurified by reverse phase HPLC using a Gilson system, eluting with10-90% acetonitrile in water containing 0.1% v/v trifluoroacetic acid.The desired fractions were combined and freeze-dried to provide thetitle compound. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm 12.84 (s,1H), 9.98 (s, 1H), 8.82 (s, 1H), 8.05 (dd, 2H), 7.79 (d, 2H), 7.70-7.53(m, 2H), 7.53-7.24 (m, 6H), 6.99 (s, 2H), 6.95 (d, 1H), 6.00 (s, 1H),4.99 (s, 2H), 4.96 (s, 2H), 4.37 (q, 2H), 4.25-4.15 (m, 2H), 3.88 (t,2H), 3.83 (s, 2H), 3.69-3.61 (m, 2H), 3.44-3.30 (m, 4H), 3.08-2.90 (m,4H), 2.90-2.72 (m, 4H), 2.27-2.04 (m, 5H), 2.04-1.89 (m, 2H), 1.77-0.94(m, 28H), 0.91-0.78 (m, 14H). MS (ESI) mr/e 1499.5 (M+H)⁺.

2.36 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N⁵-carbamoyl-N-{4-[({[2-({3-[(4-{2-carboxy-6-[8-([1,3]thiazolo[4,5-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](3-phosphonopropyl)carbamoyl}oxy)methyl]phenyl}-L-ornithinamide(Synthon IA)

The title compound was prepared by substituting Example 1.39.2 forExample 1.2.9 in Example 2.1. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δppm 9.98 (s, 1H), 8.60 (dd, 1H), 8.52 (dd. 1f), 8.06 (d, 1H), 7.78 (d,1H), 7.65 (d, 1H), 7.59 (br In, 2H), 7.50 (m, 1H), 7.45 (d, 1H), 7.38(m, 2H), 7.28 (s, 1H), 7.27 (d, 2H), 6.99 (s, 2H), 6.97 (d, 1H), 5.98(br s, 1H), 4.98 (s, 4H), 4.39 (m, 1H), 4.19 (br m, 1H), 3.88 (t, 2H),3.80 (br d, 2H), 3.36 (br m, 3H), 3.24 br (m, 4H), 2.98 (m, 4H), 2.16(m, 2H), 2.12 (s, 3H), 1.95 (br m, 1H), 1.67 (br m, 3H), 1.34-1.47 (m,9H), 1.08-1.23 (m, 1H), 0.95 (br m. 2H), 0.85-0.80 (m, 12H). MS (ESI)m/e 1465.5 (M−H)⁻.

2.37 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-carbamoyl-N-{4-[({[2-({3-[(4-{2-carboxy-6-[8-([1,3]thiazolo[5,4-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](3-phosphonopropyl)carbamoyloxy}methyl]phenyl}-L-ornithinamide(Synthon IF)

The title compound was prepared by substituting Example 1.40.2 forExample 1.2.9 in Example 2.1. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δppm 9.98 (s, 1H), 8.52 (dd, 1H), 8.16 (dd, 1H), 8.05 (br d, 1H), 7.78(br d, 1H), 7.62 (m, 1H), 7.58 (br m, 2H), 7.52 (m, 2H), 7.44 (d, 1H),7.38 (t, 1H), 7.29 (s, 1H) 7.27 (d, 2H), 6.99 (s, 2H), 6.97 (d, 1H),4.98 (s, 2H), 4.96 (s, 2H), 4.39 (m, 1H), 4.19 (br m, 1H), 3.88 (t, 2H),3.80 (br d, 2H), 3.36 (br m, 3H), 3.24 br (m, 4H), 2.98 (m, 4H), 2.16(m. 2H), 2.12 (s, 3H), 1.95 (br m, 1H), 1.67 (br m, 3H), 1.47-1.34 (m,9H), 1.08-1.23 (m, 11H), 0.95 (br m, 2H), 0.85-0.80 (m, 12H). MS (ESI)m/e 1451.5 (M−H)⁻.

2.38 Synthesis ofN-{6-[(chloroacetyl)amino]hexanoyl}-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-L-alaninamide(Synthon IG) 2.38.13-(1-((3-(2-((((4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)(2-sulfoethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid

A solution of Example 1.2.9 (0.050 g), (9H-fluoren-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxobutan-2-yl)carbamate(0.039 g) and N,N-diisopropylethylamine (0.027 mL) inN,N-dimethylformamide (1 mL) was stirred at room temperature. Afterstirring overnight, diethylamine (0.027 mL) was added to the reaction,and stirring was continued for 2 hours. The reaction was quenched withtrifluoroacetic acid, and the mixture was purified by reverse phase HPLCusing a Gilson system, eluting with 5-75% acetonitrile in watercontaining 0.1% v/v trifluoroacetic acid. The desired fractions werecombined and freeze-dried to provide the title compound. MS (ESI) m/e1499.5 (M+H)⁺.

2.38.2N-{6-[(chloroacetyl)amino]hexanoyl}-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-L-alaninamide

To a solution of 6-(2-chloroacetamido)hexanoic acid (6 mg) and2-(3H-[1.2.3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisouroniumhexafluorophosphate(V) (0.011 g) in N,N-dimethylformamide (1 mL) wasadded N,N-diisopropylethylamine (0.015 mL), and the reaction stirred for5 minutes. This solution was added to Example 2.38.1 (0.022 g) and wasstirred for 1 hour. The reaction was diluted with N,N-dimethylformamide(1 mL) and water (0.5 mL). The mixture was purified by reverse phaseHPLC using a Gilson system, eluting with 10-90% acetonitrile in watercontaining 0.1% v/v trifluoroacetic acid. The desired fractions werecombined and freeze-dried to provide the title compound. ¹H NMR (400MHz, dimethyl sulfoxide-d₆) δ ppm 12.83 (s, 1H), 9.93 (s, 1H), 8.20-8.10(m, 2H), 8.04 (d, 1H), 7.83-7.76 (m, 2H), 7.64-7.55 (m, 3H), 7.55-7.50(m, 1H) 7.50-7.41 (m, 2H), 7.40-7.32 (m, 2H), 7.32-7.24 (m, 3H), 6.96(d, 1H), 5.07-4.92 (m, 3H), 4.39 (p, 1H), 4.18 (dd, 2H), 4.01 (s, 2H),3.92-3.76 (m, 6H), 3.54-3.32 (m, 4H), 3.25 (t, 2H), 3.13-2.93 (m, 4H),2.72-2.58 (m, 2H), 2.29-2.12 (m, 2H), 2.09 (s, 3H), 2.05-1.92 (m, 1H),1.58-0.89 (m, 18H), 0.89-0.77 (m, 12H). MS (ESI) m/e 1362.2 (M+H)⁺.

2.39 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-5-(carboxymethoxy)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](methyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide(Synthon I)

The title compound was prepared by substituting Example 1.41.3 forExample 2.5.3 in Example 2.5.4. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆)δ ppm 10.03 (s, 1H), 9.96 (s, 1H), 8.26-8.34 (m, 1H), 7.95-8.11 (m, 2H),7.73-7.82 (m, 2H), 7.22-7.70 (m, 11H), 6.95-7.05 (m, 3H), 6.89 (d, 1H),5.23 (s, 1H), 4.98 (d, 3H), 4.83 (s, 1H), 4.33-4.43 (m, 1H), 4.11-4.23(m, 1H), 3.74-3.95 (m, 3H), 3.22-3.39 (m, 10H), 2.78-3.06 (m, 12H),1.91-2.22 (m, 8H), 0.93-1.68 (m, 20H), 0.77-0.88 (m, 10H). MS (ESI) m/e1432.2 (M+H)⁺.

2.40 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-[4-({[(2-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)amino}ethyl)(2-carboxyethyl)carbamoyl]oxy}methyl)phenyl]-N⁵-carbamoyl-L-ornithinamide(Synthon IJ)

The title compound was prepared as described in Example 2.1, replacingExample 1.2.9 with Example 1.38.2. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 12.86 (s, 1H), 9.99 (s, 1H), 9.10 (s, 1H), 8.04 (t,2H), 7.73-7.85 (m, 2H), 7.61 (t, 3H), 7.41-7.55 (m, 3H), 7.26-7.39 (m,5H), 6.99 (s, 2H), 6.95 (d, 1H), 6.00 (s, 1H), 4.99 (d, 4H), 4.34-4.45(m, 2H), 4.19 (dd, 2H), 3.88 (t, 2H), 3.82 (s, 2H), 3.36 (t, 4H),2.85-3.09 (m, 5H), 2.06-2.22 (m, 4H), 1.89-2.02 (m, 1H), 0.94-1.77 (m,20H), 0.77-0.90 (m, 11H). MS (ESI) m/e 1567.4 (M+H)⁺.

2.41 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-[1-({3-[2-({(2S)-2-[{[(4-([(2S)-5-(carbamoylamino)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}pentanoyl]amino}benzyl)oxy]carbonyl)(2-carboxyethyl)amino]-3-carboxypropanoyl}amino)ethoxy]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylicAcid (Synthon IK)

The title compound was prepared as described in Example 2.1, replacingExample 1.2.9 with Example 1.32.4. MS (ESI) m/e 1592.4 (M−H)⁻.

2.42 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(H)-yl]-3-(1-{[3-(2-{[(2S)-2-(([(4-{[(2S)-5-(carbamoylamino)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}pentanoyl]amino}benzyl)oxy]carbonyl)amino)-3-carboxypropanoyl](2-sulfoethyl)amino}ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylicAcid (Synthon IL)

The title compound was prepared as described in Example 2.1, replacingExample 1.2.9 with Example 1.44.2. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 12.82 (s, 1H), 9.96 (s, 1H). 8.03 (t, 2H), 7.77 (d,2H), 7.39-7.62 (m, 7H), 7.30-7.39 (m, 2H), 7.22-7.29 (m, 3H), 6.98 (s,2H), 6.92-6.96 (m, 1H), 5.97 (s, 1H), 4.83-5.05 (m, 3H), 3.83-3.92 (m,1H), 3.79 (s, 1H), 3.00 (s, 2H), 2.03-2.22 (m, 8H), 1.94 (s, 2H), 1.34(d, 30H), 0.69-0.90 (m, 13H). MS (ESI) m/e 1565.5 (M−H)⁻.

2.43 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-[4-({[(4-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](3-carboxypropyl)amino}piperidin-1-yl)carbonyl]oxy}methyl)phenyl]-N⁵-carbamoyl-L-ornithinamide(Synthon IM)

A solution of Example 1.42.2 (0.045 g),4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl) carbonate (0.035 g) and N,N-diisopropylethylamine (0.038mL) in N,N-dimethylformamide (0.5 mL) was stirred at room temperature.After stirring for 3 hours, the reaction was diluted withN,N-dimethylformamide (1.25 mL) and water (0.5 mL). The mixture waspurified by reverse phase HPLC using a Gilson system, eluting with10-90% acetonitrile in water containing 0.1% v/v trifluoroacetic acid.The desired fractions were combined and freeze-dried to provide thetitle compound. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm 12.76 (s,1H), 9.91 (s, 1H), 8.79 (s, 1H), 7.98 (dd 2H), 7.72 (d, 2H), 7.68-7.47(m, 3H), 7.47-7.00 (m, 7H), 6.96-6.83 (m, 3H), 5.93 (s, 1H), 4.91 (d,3H), 4.30 (q, 1H), 4.17-3.97 (m, 4H), 3.96-3.53 (m, 4H), 3.34-2.65 (m,12H), 2.25 (t, 2H), 2.16-1.67 (m, 12H), 1.67-0.88 (m, 26H), 0.84-0.70(m, 12H). MS (ESI) m/e 1513.6 (M+H)⁺.

2.44 Synthesis of4-[(1E)-3-({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-5-(carboxymethoxy)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](methyl)carbamoyl}oxy)prop-1-en-1-yl]-2-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-beta-alanyl}amino)phenylbeta-D-glucopyranosidumnic Acid (Synthon IO) 2.44.1(E)-tert-butyldimethyl((3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)allyl)oxy)silane

To a flask charged with tert-butyldimethyl(prop-2-yn-1-yloxy)silane (5g) and dichloromethane (14.7 mL) under nitrogen atmosphere was addeddropwise 4,4,5,5-tetramethyl-1,3,2-dioxaborolane (3.94 g). The mixturewas stirred at room temperature for one minute then transferred viacannula to a nitrogen-sparged flask containing Cp₂ZrClH(chloridobis(η5-cyclopentadienyl)hydridozirconium, Schwartz's Reagent)(379 mg). The resulting reaction mixture was stirred at room temperaturefor 16 hours. The mixture was carefully quenched with water (15 mL), andthen extracted with diethyl ether (3×30 mL). The combined organic phaseswere washed with water (15 mL), dried over MgSO₄, filtered, and purifiedby silica gel chromatography, eluting with a gradient from 0-8% ethylacetate/heptanes to give the title compound. MS (ESI) m/z 316.0(M+NH₄)⁺.

2.44.2(2S,3R,4S,6S)-2-(4-bromo-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate

(2R,3R,4S,5S,6S)-2-Bromo-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (5 g) was dissolved in acetonitrile (100 mL). Ag₂O (2.92 g)was added to the solution, and the reaction was stirred for 5 minutes atroom temperature. 4-Bromo-2-nitrophenol (2.74 g) was added, and thereaction mixture was stirred at room temperature for 4 hours. The silversalt residue was filtered through diatomaceous earth, and the filtratewas concentrated under reduced pressure. The residue was purified bysilica gel chromatography, eluting with a gradient of 10-70% ethylacetate in heptanes, to give the title compound. MS (ESI+) m/z 550.9(M+NH₄)⁺.

2.44.3(2S,3R,4S,5S,6S)-2-(4-((E)-3-((tert-butyldimethylsilyl)oxy)prop-1-en-1-yl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate

Example 2.44.2 (1 g), sodium carbonate (0.595 g),tris(dibenzylideneacetone)dipalladium (Pd₂(dba)₃) (0.086 g), and1,3,5,7-tetramethyl-6-phenyl-2,4,8-trioxa-6-phosphaadamantane (0.055 g)were combined in a 3-neck 50-mL round bottom flask equipped with areflux condenser and the system was degassed with nitrogen. Separately,a solution of Example 2.44.1 (0.726 g) in tetrahydrofuran (15 mL) wasdegassed with nitrogen for 30 minutes. The latter solution wastransferred via cannula into the flask containing the solid reagents,followed by addition of degassed water (3 mL) via syringe. The reactionwas heated to 60° C. for two hours. The reaction mixture was partitionedbetween ethyl acetate (3×30 mL) and water (30 mL). The combined organicphases were dried (Na₂SO₄), filtered, and concentrated. The residue waspurified by silica gel chromatography, eluting with a gradient from0-35% ethyl acetate in heptanes, to provide the title compound. MS(ESI+) m/z 643.1 (M+NH₄)⁺.

2.44.4(2S,3R,4S,5S,6S)-2-(2-amino-4-((E)-3-hydroxyprop-1-en-1-yl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate

A 500-mL three-neck, nitrogen-flushed flask equipped with apressure-equalizing addition funnel was charged with zinc dust (8.77 g).A degassed solution of Example 2.44.3 (8.39 g) in tetrahydrofuran (67mL) was added via cannula. The resulting suspension was chilled in anice bath, and 6N HCl (22.3 mL) was added dropwise via the additionfunnel at such a rate that the internal temperature of the reaction didnot exceed 35° C. After the addition was complete, the reaction wasstirred for two hours at room temperature, and filtered through a pad ofdiatomaceous earth, rinsing with water and ethyl acetate. The filtratewas treated with saturated aqueous NaHCO₃ solution until the water layerwas no longer acidic, and the mixture was filtered to remove theresulting solids. The filtrate was transferred to a separatory funnel,and the layers were separated. The aqueous layer was extracted withethyl acetate (3×75 mL), and the combined organic layers were washedwith water (100 mL), dried over Na₂SO₄, filtered, and concentrated. Theresidue was triturated with diethyl ether and the solid collected byfiltration to provide the title compound. MS (ESI+) m/z 482.0 (M+H)⁺.

2.44.5 (9H-fluoren-9-yl)methyl (3-chloro-3-oxopropyl)carbamate

To a solution of 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoicacid (5.0 g) in dichloromethane (53.5 mL) was added sulfurous dichloride(0.703 mL). The mixture was stirred at 60° C. for one hour. The mixturewas cooled and concentrated to give the title compound, which was usedin the next step without further purification.

2.44.6(2S,3R,4S,5S,6S)-2-(2-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-((E)-3-hydroxyprop-1-en-1-yl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate

Example 2.44.4 (6.78 g) was dissolved in dichloromethane (50 mL), andthe solution was chilled to 0° C. in an ice bath.N,N-Diisopropylethylamine (3.64 g) was added, followed by dropwiseaddition of a solution of Example 2.44.5 (4.88 g) in dichloromethane (50mL). The reaction was stirred for 16 hours allowing the ice bath to cometo room temperature. Saturated aqueous NaHCO₃ solution (100 mL) wasadded, and the layers were separated. The aqueous layer was furtherextracted with dichloromethane (2×50 mL). The extracts were dried overNa₂SO₄, filtered, concentrated and purified by silica gelchromatography, eluting with a gradient of 5-95% ethyl acetate/heptane,to give an inseparable mixture of starting aniline and desired product.The mixture was partitioned between 1N aqueous HCl (40 mL) and a 1:1mixture of diethyl ether and ethyl acetate (40 mL), and then the aqueousphase was further extracted with ethyl acetate (2×25 mL). The organicphases were combined, washed with water (2×25 mL), dried over Na₂SO₄,filtered, and concentrated to give the title compound. MS (ESI+) m/z774.9 (M+H)⁺.

2.44.7(2S,3R,4S,5S,6S)-2-(2-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-((E)-3-(((4-nitrophenoxy)carbonyl)oxy)prop-1-en-1-yl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate

Example 2.44.6 (3.57 g) was dissolved in dichloromethane (45 mL) andbis(4-nitrophenyl)carbonate (2.80 g) was added, followed by dropwiseaddition of N,N-diisopropylethylamine (0.896 g). The reaction mixturewas stirred at room temperature for two hours. Silica gel (20 g) wasadded to the reaction solution, and the mixture was concentrated todryness under reduced pressure, keeping the bath temperature at or below25° C. The silica residue was loaded atop a column, and the product waspurified by silica gel chromatography, eluting with a gradient from0-100% ethyl acetate-heptane, providing partially purified product whichwas contaminated with nitrophenol. The material was triturated withmethyl tert-butyl ether (250 mL), and the resulting slurry was allowedto sit for 1 hour. The product was collected by filtration. Threesuccessive crops were collected in a similar fashion to give the titlecompound. MS (ESI+) m/z 939.8 (M+H)⁺.

2.44.83-(1-((3-(2-(((((E)-3-(3-(3-aminopropanamido)-4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)phenyl)allyl)oxy)carbonyl)(methyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-5-(carboxymethoxy)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid

To a cold (0° C.) solution of Example 2.44.7 (19.7 mg) and Example1.41.3 (18.5 mg) in N,N-dimethylformamide (2 mL) was addedN,N-diisopropylethylamine (0.054 mL). The reaction was slowly warmed toroom temperature and stirred overnight. To the reaction mixture wasadded water (2 mL) and lithium hydroxide monohydrate (50 mg), and themixture was stirred overnight. The mixture was acidified withtrifluoroacetic acid and filtered. The mixture was purified by reversephase HPLC (Gilson system), eluting with 10-85% acetonitrile in 0.1%trifluoroacetic acid in water, to provide the title compound. MS (ESI)m/e 1273.2 (M+H)⁺.

2.44.94-[(1E)-3-({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-5-(carboxymethoxy)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](methyl)carbamoyl}oxy)prop-1-en-1-yl]-2-((N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-beta-alanyl)amino)phenylbeta-D-glucopyranosiduronic Acid

To a solution of Example 2.44.8 (10 mg) and 2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (2.3 mg) inN,N-dimethylformamide (2 mL) was added N,N-diisopropylethylamine (0.054mL). The reaction was stirred overnight. The reaction mixture wasdiluted with methanol (2 mL) and acidified with trifluoroacetic acid.The mixture was purified by reverse phase HPLC (Gilson system), elutingwith 10-85% acetonitrile in 0.1% trifluoroacetic acid in water, to givethe title compound. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm 12.70(s, 1H), 9.03 (s, 1H), 8.25 (s, 1H), 8.01 (d, 1H), 7.87 (t, 1H), 7.77(d, 1H), 7.69 (d, 1H), 7.41-7.55 (m, 2H), 7.23-7.38 (m, 2H), 6.79-7.16(m, 7H), 6.56 (d, 1H), 6.09-6.25 (m, 1H), 4.96-5.07 (m, 3H), 4.84 (s,3H), 4.64 (d, 3H), 3.87-3.97 (m, 5H), 3.24-3.47 (m, 12H), 2.77-2.95 (m,6H), 1.94-2.08 (m, 6H), 0.92-1.56 (m, 20H), 0.74-0.86 (m, 6H). MS (ESI)m/e 1487.3 (M+Na)⁺.

2.45 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide(Synthon IP)

The title compound was prepared by substituting Example 1.43.7 forExample 2.5.3 in Example 2.5.4. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆)δ ppm 13.09 (s, 1H), 9.99 (s, 1H), 9.02 (s, 1H), 8.30-8.40 (m, 3H),7.93-8.25 (m, 6H), 7.23-7.86 (m, 10H), 6.92-7.05 (m, 2H), 4.99 (d, 2H),4.36-4.44 (m, 2H), 4.14-4.23 (m, 2H), 2.87-3.35 (m, 12H), 2.81 (t, 2H),2.59-2.70 (m, 2H), 1.84-2.28 (m, 8H), 0.97-1.77 (m, 20H), 0.77-0.88 (m,10H). MS (ESI) m/e 1448.3 (M+Na)⁺.

2.46 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-[4-({[(2-{[8-(1,3-benzothiazol-2-ylcarbamoyl)-2-(6-carboxy-5-{1-[(3,5-dimethyl-7-{2-[methyl(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridin-2-yl)-1,2,3,4-tetrahydroisoquinolin-5-yl]oxy}ethyl)carbamoyl]oxy}methyl)phenyl]-N⁵-carbamoyl-L-ornithinamide(Synthon IS)

The title compound was prepared as described in Example 2.1, replacingExample 1.2.9 with Example 1.46.2. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 12.69 (s, 1H), 9.97 (s, 1H), 8.97 (s, 1H), 8.04 (dd,2H), 7.78 (d, 2H), 7.71 (d, 1H), 7.59 (d, 2H), 7.44-7.54 (m, 3H),7.26-7.37 (m, 4H), 6.96-7.03 (m, 4H), 5.97 (s, 1H), 4.99 (d, 4H),4.31-4.45 (m, 1H), 4.18 (dd, 1H), 4.09 (s, 2H), 3.85-3.93 (m, 2H), 3.83(s, 2H), 3.39-3.47 (m, 2H), 3.24-3.39 (m, 4H), 3.12-3.24 (m, 2H),2.75-3.07 (m, 9H), 2.06-2.23 (m, 5H), 1.90-2.01 (m, 1H), 1.54-1.75 (m,2H), 1.24-1.52 (m, 12H), 0.91-1.24 (m, 8H), 0.77-0.88 (m, 12H). MS (ESI)m/e 1525.4 (M+H)⁺.

2.47 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-[4-({[(2-{[8-(1,3-benzothiazol-2-ylcarbamoyl)-2-(6-carboxy-5-(1-[(3,5-dimethyl-7-{2-[methyl(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-ylpyridin-2-yl)-1,2,3,4-tetrahydroisoquinolin-5-yl]oxy}ethyl)(2-sulfoethyl)carbamoyl]oxy}methyl)phenyl]-N⁵-carbamoyl-L-ornithinamide(Synthon IU)

The title compound was prepared as described in Example 2.1, replacingExample 1.2.9 with Example 1.47.2. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 12.70 (s, 1H), 9.99 (s, 1H), 8.97 (s, 1H), 8.04 (dd,2H), 7.78 (d, 2H), 7.71 (d, 1H), 7.59 (d, 2H), 7.43-7.55 (m, 2H),7.28-7.37 (m, 4H), 6.94-7.07 (m, 4H), 6.05 (s, 1H), 4.93-5.11 (m, 4H),4.31-4.46 (m, 2H), 4.12-4.26 (m, 4H), 3.80-3.95 (m, 4H), 3.40-3.50 (m,2H), 3.24-3.40 (m, 6H), 3.13-3.24 (m, 2H), 2.74-3.08 (m, 9H), 2.63-2.73(m, 2H), 2.05-2.23 (m, 5H), 1.96 (s, 1H), 1.52-1.77 (m, 2H), 1.23-1.53(m, 12H), 0.97-1.22 (m, 8H), 0.77-0.89 (m, 12H). MS (ESI) m/e 1631.5(M−H)⁻.

2.48 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-[4-({[(2-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)amino}ethyl)(2-sulfoethyl)carbamoyl]oxy}methyl)phenyl]-N³-carbamoyl-L-ornithinamide(Synthon IV)

The title compound was prepared as described in Example 2.1, replacingExample 1.2.9 with Example 1.48.2. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 12.82 (s, 1H), 10.00 (s, 1H), 9.29-9.57 (m, 1H),8.05 (t, 2H), 7.79 (d, 2H), 7.51-7.63 (m, 4H), 7.40-7.50 (m, 2H),7.27-7.39 (m. 5H), 6.93-7.02 (m, 3H), 4.99 (d, 3H), 4.30-4.47 (m, 1H),4.19 (t, 1H), 3.79-3.92 (m, 3H), 3.60-3.74 (m, 2H), 3.01 (s, 9H), 2.70(d, 4H), 2.05-2.23 (m, 6H), 1.96 (d, 2H), 1.53-1.78 (m, 3H), 1.22-1.54(m, 13H), 0.89-1.22 (m, 9H), 0.75-0.89 (m, 13H). MS (ESI) m/e 1603.3(M+H)⁺.

2.49 Synthesis ofN-{6-[(chloroacetyl)amino]hexanoyl}-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide(Synthon IZ) 2.49.13-(1-(((1r,3r)-3-(2-((((4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)(2-sulfoethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid

A solution of Example 1.2.9 (0.045 g) (9H-fluoren-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-1-oxobutan-2-yl)carbamate(0.043 g) and N,N-diisopropylethylamine (0.041 mL) were stirred togetherin N,N-dimethylformamide (1 mL) at room temperature. After stirringovernight, diethylamine (0.024 mL) was added to the reaction, andstirring was continued for 2 hours. The reaction was quenched withtrifluoroacetic acid then purified by reverse phase HPLC using a Gilsonsystem, eluting with 10-75% acetonitrile in water containing 0.1% v/vtrifluoroacetic acid. The desired fractions were combined andfreeze-dried to provide the title compound.

2.49.2N-{6-[(chloroacetyl)amino]hexanoyl}-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydriosoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N-carbamoyl-1-ornithinamide

A solution of 6-(2-chloroacetamido)hexanoic acid (6.43 mg) and2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisouroniumhexafluorophosphate(V) (0.012 g) in N,N-dimethylformamide (0.5 mL) wasadded N,N-diisopropylethylamine (0.019 mL), and the reaction stirred for5 minutes. This solution was added to Example 2.49.1 (0.026 g) and wasstirred for 1 hour. The reaction was diluted with N,N-dimethylformamide(1 mL) and water (0.5 mL). The mixture was purified by reverse phaseHPLC using a Gilson system, eluting with 10-60% acetonitrile in watercontaining 0.1% v/v trifluoroacetic acid. The desired fractions werecombined and freeze-dried to provide the title compound. ¹H NMR (500MHz, dimethyl sulfoxide-d₆) δ ppm 12.85 (s, 1H), 9.99 (s, 1H), 8.18 (q,1H), 8.08 (d, 1H), 8.04 (d, 1H), 7.84-7.76 (m, 2H), 7.64-7.56 (m, 3H),7.56-7.50 (m, 1H), 7.47 (t, 1H), 7.43 (d, 1H), 7.37 (d, 1H), 7.35 (d,1H), 7.29 (s, 1H), 7.27 (d, 2H), 6.95 (d, 1H), 6.05 (s, 1H), 5.05-4.91(m, 4H), 4.48-4.33 (m, 1H), 4.26-4.14 (m, 1H), 4.02 (s, 2H), 3.88 (t,2H), 3.81 (d, 2H), 3.25 (t, 2H), 3.14-2.98 (m, 6H), 2.98-2.87 (m, 2H),2.74-2.59 (m, 2H), 2.27-2.05 (m, 6H), 2.04-1.92 (m, 1H), 1.78-1.65 (m,1H), 1.65-1.53 (m, 1H), 1.53-0.90 (m, 22H), 0.90-0.73 (m, 12H). MS (ESI)m/e 1448.2 (M+H)⁺.

2.50 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[4-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydro-2H-1,4-benzoxazin-6-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide(Synthon JD)

The title compound was prepared by substituting Example 1.51.8 forExample 2.5.3 in Example 2.5.4. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆)δ ppm 9.56 (s, 1H), 8.51-8.59 (m, 1H), 7.89 (d, 1H), 7.82 (d, 1H),7.69-7.77 (m, 2H), 7.34-7.62 (m, 7H), 7.16-7.34 (m, 4H), 6.95 (dd, 1H),5.95-6.05 (m, 1H), 4.95 (s, 2H), 4.06-4.44 (m, 6H), 3.85 (s, 3H),3.39-3.59 (m, 7H), 2.61-2.74 (m, 3H), 2.19 (s, 3H), 1.88-2.16 (m, 3H)0.96-1.75 (m, 22H), 0.71-0.89 (m, 13H). MS (ESI) m/e 1454.2 (M+Na)⁺.

2.51 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-[4-({[(2-{[8-(1,3-benzothiazol-2-ylcarbamoyl)-2-(6-carboxy-5-{1-[(3,5-dimethyl-7-{2-[methyl(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-yl)methy]-5-methyl-1H-pyrazol-4-yl}pyridin-2-yl)-1,2,3,4-tetrahydroisoquinolin-5-yl]oxy}ethyl)(2-carboxyethyl)carbamoyl]oxy}methyl)phenyl]-N⁵-carbamoyl-L-ornithinamide(Synthon J)

The title compound was prepared as described in Example 2.1, replacingExample 1.2.9 with Example 1.49.2. ¹H NMR (500 MHz, dimethylsulfoxide-d₆) δ ppm 12.71 (s, 1H), 10.00 (s, 1H), 8.97 (s, 1H), 8.08 (d,1H), 8.02 (d, 1H), 7.78 (d, 2), 7.72 (d, 1H), 7.60 (d, 2H), 7.52 (d,1H), 7.44-7.50 (m, 1H), 7.27-7.39 (m, 4H), 6.96-7.06 (m, 3H), 5.98 (s,1H), 5.01 (d, 4H), 4.31-4.46 (m, 1H), 4.18 (s, 3H), 3.79-3.95 (m, 4H),3.67-3.76 (m, 2H), 3.12-3.39 (m, 6H), 2.73-3.07 (m, 8H), 2.04-2.24 (m,4H), 1.87-2.02 (m, 1H), 1.22-1.75 (m, 12H), 0.96-1.20 (m, 7H), 0.76-0.90(m, 10H). MS (ESI) m/e 1597.4 (M+H)⁺.

2.52 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-5-(3-sulfopropoxy)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](methyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide(Synthon JK)

The title compound was prepared by substituting Example 1.52.4 forExample 2.5.3 in Example 2.5.4. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆)δ ppm 9.97 (s, 1H), 7.96-8.11 (m, 2H), 7.67-7.82 (m, 3H), 7.59 (d, 2H),7.42-7.52 (m, 2H), 7.23-7.36 (m, 4H), 6.91-7.08 (m, 4H), 4.99 (d, 4H),4.33-4.47 (m, 1H), 4.14-4.23 (m, 4H), 3.86-3.95 (m, 6H), 3.21-3.45 (m,15H), 2.75-3.07 (m, 9H), 2.56-2.69 (m, 2H), 1.93-2.20 (In, 8H),0.88-1.72 (m, 20f), 0.74-0.89 (m, 11H). MS (ESI) m/e 1496.3 (M+Na)⁺.

2.53 Synthesis ofN-[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide(Synthon JJ)

A solution of Example 2.49.1 (0.030 g), 2,5-dioxopyrrolidin-1-yl3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate (6.34 mg) andN,N-diisopropylethylamine (0.012 mL) in N,N-dimethylformamide (0.5 mL)was stirred at room temperature. After 1 hour the reaction was quenchedwith a 3:1 mixture of N,N-dimethylformamide:water (1.5 mL). The mixturewas purified by reverse phase HPLC using a Gilson system, eluting with10-85% acetonitrile in water containing 0.1% v/v trifluoroacetic acid.The desired fractions were combined and freeze-dried to provide thetitle compound. ¹H NMR (500 MHz, dimethyl sulfoxide-d₆) δ ppm 12.85 (s,1H), 9.99 (s, 1H), 8.18 (q, 1H), 8.12-8.00 (m, 2H), 7.86-7.75 (m, 2H),7.65-7.55 (m, 3H), 7.53 (dd, 1H), 7.47 (t, 1H), 7.43 (d, 1H), 7.36 (q,2H), 7.33-7.23 (m, 3H), 6.95 (d, 1H), 6.05 (s, 1H), 5.03-4.92 (m, 4H),4.39 (q, 1H), 4.24-4.14 (m, 1H), 4.02 (s, 2H), 3.88 (t, 2H), 3.81 (d,2H), 3.39-3.16 (m, 2H), 3.14-2.86 (m, 10H), 2.68-2.60 (m, 2H), 2.25-2.04(m, 6H), 2.03-1.90 (m, 1H), 1.78-1.65 (m, 1H), 1.64-1.54 (m, 1H),1.54-0.90 (m, 20H), 0.89-0.75 (m, 12H). MS (ESI) m/e 1410.1 (M+H)⁺.

2.54 Synthesis ofN-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide(Synthon JL)

A solution of Example 2.49.1 (0.039 g). 2,5-dioxopyrrolidin-1-yl2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (7.81 mg) andN,N-diisopropylethylamine (0.016 mL) in N,N-dimethylformamide (0.5 mL)was stirred at room temperature. After 1 hour, the reaction was quenchedwith a 3:1 mixture of N,N-dimethylformamide:water (1.5 mL). The mixturewas purified by reverse phase HPLC using a Gilson system, eluting with10-85% acetonitrile in water containing 0.1% v/v trifluoroacetic acid.The desired fractions were combined and freeze-dried to provide thetitle compound. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm 12.85 (s,1H), 10.00 (d, 1H), 8.24 (d, 2H), 8.04 (d, 1H), 7.79 (d, 1H), 7.59 (q,3H), 7.53 (dd, 1H), 7.47 (1, 1H), 7.43 (d, 1H), 7.36 (td, 2H), 7.30 (s,1H), 7.27 (d, 2H), 7.07 (s, 2H), 6.96 (d, 1H), 5.04-4.85 (m, 4H), 4.39(q, 2H), 4.26 (dd, 2H), 4.13 (s, 2H), 3.86-3.17 (m, 8H), 3.07-2.81 (m,4H), 2.63 (t, 2H), 2.09 (s, 3H), 2.03-1.79 (m, 1H), 1.75-1.51 (m, 2H),1.51-1.03 (m, 12H), 1.01-0.76 (m, 16H). MS (ESI) m/e 1394.4 (M−H)⁻.

2.55 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3-(2-([(2S)-2-({[(4-{[(2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl]oxy}-3-[(3-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}propanoyl)amino]benzyl)oxy]carbonyl}amino)-3-sulfopropanoyl](methyl)amino}ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylicAcid (Synthon FE) 2.55.1(2S,3R,4S,5S,6S)-2-(4-formyl-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate

To a solution of(2R,3R,4S,5S,6S)-2-bromo-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (4 g) in acetonitrile (100 mL)) was added silver(I) oxide(10.04 g) and 4-hydroxy-3-nitrobenzaldehyde (1.683 g). The reactionmixture was stirred for 4 hours at room temperature and filtered. Thefiltrate was concentrated, and the residue was purified by silica gelchromatography, eluting with 5-50% ethyl acetate in heptanes, to providethe title compound. MS (ESI) me (M+18)⁺.

2.55.2(2S,3R,4S,5S,6S)-2-(4-(hydroxymethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate

To a solution of Example 2.55.1 (6 g) in a mixture of chloroform (75 mL)and isopropanol (18.75 mL) was added 0.87 g of silica gel. The resultingmixture was cooled to 0° C. NaBH₄ (0.470 g) was added, and the resultingsuspension was stirred at 0° C. for 45 minutes. The reaction mixture wasdiluted with dichloromethane (100 mL) and filtered through diatomaceousearth. The filtrate was washed with water and brine and concentrated togive the crude product, which was used without further purification. MS(ESI) m/e (M+NH₄)⁺:

2.55.3(2S,3R,4S,5S,6S)-2-(2-amino-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate

A stirred solution of Example 2.55.2 (7 g) in ethyl acetate (81 mL) washydrogenated at 20° C. under 1 atmosphere H₂, using 10% Pd/C (1.535 g)as a catalyst for 12 hours. The reaction mixture was filtered throughdiatomaceous earth, and the solvent was evaporated under reducedpressure. The residue was purified by silica gel chromatography, elutingwith 95/5 dichloromethane/methanol, to give the title compound.

2.55.4 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoic Acid

3-Aminopropanoic acid (4.99 g) was dissolved in 10% aqueous Na₂CO₃solution (120 mL) in a 500 mL flask and cooled with an ice bath. To theresulting solution. (9H-fluoren-9-yl)methyl carbonochloridate (14.5 g)in 1,4-dioxane (100 mL) was gradually added. The reaction mixture wasstirred at room temperature for 4 hours, and water (800 mL) was thenadded. The aqueous phase layer was separated from the reaction mixtureand washed with diethyl ether (3×750 mL). The aqueous layer wasacidified with 2N HCl aqueous solution to a pH value of 2 and extractedwith ethyl acetate (3×750 mL). The organic layers were combined andconcentrated to obtain crude product. The crude product wasrecrystallized in a mixed solvent of ethyl acetate: hexane 1:2 (300 mL)to give the title compound.

2.55.5 (9H-fluoren-9-yl)methyl (3-chloro-3-oxopropyl)carbamate

To a solution of Example 2.55.4 in dichloromethane (160 mL) was addedsulfurous dichloride (50 mL). The mixture was stirred at 60° C. for 1hour. The mixture was cooled and concentrated to give the titlecompound.

2.55.6(2S,3R,4S,5S,6S)-2-(2-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate

To a solution of Example 2.55.3 (6 g) in dichloromethane (480 mL) wasadded N,N-diisopropylethylamine (4.60 mL). Example 2.55.5 (5.34 g) wasadded, and the mixture was stirred at room temperature for 30 minutes.The mixture was poured into saturated aqueous sodium bicarbonate and wasextracted with ethyl acetate. The combined extracts were washed withwater and brine and were dried over sodium sulfate. Filtration andconcentration gave a residue that was purified via radialchromatography, using 0-100% ethyl acetate in petroleum ether as mobilephase, to give the title compound.

2.55.7(2S,3R,4S,5S,6S)-2-(2-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate

To a mixture of Example 2.55.6 (5.1 g) in N,N-dimethylformamide (200 mL)was added bis(4-nitrophenyl) carbonate (4.14 g) andN,N-diisopropylethylamine (1.784 mL). The mixture was stirred for 16hours at room temperature and concentrated under reduced pressure. Thecrude material was dissolved in dichloromethane and aspirated directlyonto a 1 mm radial Chromatotron plate and eluted with 50-100% ethylacetate in hexanes to give the title compound. MS (ESI) m/e (M+H)⁺.

2.55.83-(1-((3-(2-((R)-2-((((3-(3-aminopropanamido)-4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)amino)-N-methyl-3-sulfopropanamido)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid

A solution of Example 1.13.7 (0.055 g) and Example 2.55.7 (0.055 g) werestirred together in N,N-dimethylformamide (1.5 mL) andN,N-diisopropylethylamine (0.053 mL) was added. After stirring for 3hours, the reaction was diluted with ethyl acetate (75 mL) and washedwith water (20 mL) and brine (25 mL), dried over magnesium sulfate,filtered, and concentrated. The residue was dissolved in methanol (1 mL)and treated with lithium hydroxide hydrate (0.025 g) in water (0.6 mL).After stirring for 2 hours, the reaction was quenched withtrifluoroacetic acid (0.047 ml) and diluted with N,N-dimethylformamide(1 mL). The mixture was purified by reverse phase HPLC using a Gilsonsystem, eluting with 10-80% acetonitrile in water containing 0.1% v/vtrifluoroacetic acid. The desired fractions were combined andfreeze-dried to provide the title compound as a trifluoroacetic acidsalt.

2.55.96-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3-(2-{[(2S)-2-({[(4-{[(2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl]oxy}-3-[(3-{([6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}propanoyl)amino]benzyl)oxy]carbonyl}amino)-3-sulfopropanoyl](methyl)amino}ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylicAcid

A solution of Example 2.55.8 (0.013 g) and 2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (3.07 mg) were stirredin N,N-dimethylformamide (1 mL) and N,N-diisopropylethylamine (7.90 μL)was added. The reaction was stirred for 1 hour and diluted withN,N-dimethylformamide and water. The mixture was purified by reversephase HPLC using a Gilson system, eluting with 10-75% acetonitrile inwater containing 0.1% v/v trifluoroacetic acid. The desired fractionswere combined and freeze-dried to provide the title compound. ¹H NMR(400 MHz, dimethyl sulfoxide-d₆) δ ppm 12.84 (s, 1H), 9.07 (s, 1H), 8.15(s, 1H), 8.04 (d, 1H), 7.89 (t, 1H), 7.79 (d, 1H), 7.61 (d, 1H),7.56-7.50 (m, 1H), 7.47 (t, 1H), 7.43 (d, 1H), 7.39-7.32 (m, 2H), 7.31(s, 1H), 7.28 (d, 1H), 7.06 (d, 1H), 7.04-6.92 (m, 4H), 5.00-4.79 (m,5H), 4.73-4.64 (m, 1H), 3.94-3.78 (m, 4H), 3.57-2.84 (m, 12H), 2.84-2.56(m, 6H), 2.14-1.73 (m, 5H), 1.57-0.89 (m, 22H), 0.84 (s, 6H). MS (ESI)m/e 1516.2 (M−H)⁻.

2.56 Synthesis of4-[(1E)-3-({[2-({3-[(4-{2-carboxy-6-[8-([1,3]thiazolo[,4-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)prop-1-en-1-yl]-2-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-beta-alanyl}amino)phenylbeta-D-glucopyranosiduronic Acid (Synthon GG) 2.56.13-(1-((3-(2-(((((E)-3-(3-(3-aminopropanamido)-4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)phenyl)allyl)oxy)carbonyl)(2-sulfoethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(thiazolo[5,4-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid

Example 1.22.5 (48 mg) was dissolved in dimethylformamide (0.5 mL), andExample 2.44.7 (55 mg) and N,N-diisopropylethylamine (90 μL) were added.The reaction mixture was stirred at room temperature overnight. Thereaction was concentrated, and the residue was dissolved in methanol (1mL) and 1.94N aqueous LiOH (0.27 mL) was added. The mixture was stirredat room temperature for one hour. Purification of the mixture by reversephase chromatography (C18 column), eluting with 10-90% acetonitrile inwater containing 0.1% v/v trifluoroacetic acid provided the titlecompound as a trifluoroacetic acid salt. MS (ESI−) m/e 1291.4 (M−H)⁻.

2.56.24-[(1E)-3-({[2-({3-[(4-{2-carboxy-6-[8-([1,3]thiazolo[5,4-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)prop-1-en-1-yl]-2-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-beta-alanyl}amino)phenylbeta-D-glucopyranosiduronic Acid

The title compound was prepared by substituting Example 1.56.1 forExample 1.2.9 in Example 2.1. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δppm 13.00 (v br s, 1H), 9.03 (s, 1H), 8.53 (dd, 1H), 8.24 (s, 1H), 8.16(dd, 1H), 7.90 (br s, 1H), 7.61 (d, 1H), 7.54 (d, 1H) 7.52 (d, 1H), 7.44(d, 1H), 7.37 (t, 1H), 7.30 (s, 1H), 7.11 (br d, 1H), 7.03 (d, 1H), 6.98(s, 2H), 6.97 (d, 1H), 6.58 (m, 1H), 6.15 (m, 1H), 4.96 (s, 2H), 4.88(br m, 1H), 4.64 (br m, 2H), 3.88 (m, 3H), 3.79 (br m, 2H), 3.27-3.48(m, 14H), 3.01 (m, 2H), 2.67 (br m, 2H), 2.54 (m, 2H), 2.09 (s, 3H),2.03 (t, 2H), 1.45 (m, 6H), 1.37 (br m, 2H), 1.28-0.90 (m, 10H),0.77-0.82 (m, 6H). MS (ESI) m/e 1484.4 (M−H)⁻.

2.57 Synthesis of4-[(1E)-3-({[2-({3-[(4-{2-carboxy-6-[8-([1,3]thiazolo[4,5-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)prop-1-en-1-yl]-2-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-beta-alanyl}amino)phenylbeta-D-glucopyranosiduronic Acid (Synthon GM) 2.57.13-(1-((3-(2-(((((E)-3-(3-(3-aminopropanamido)-4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)phenyl)allyl)oxy)carbonyl)(2-sulfoethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(thiazolo[4,5-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid

The title compound was prepared by substituting Example 1.23.4 forExample 1.22.5 in Example 2.56.1. MS (ESI) m/e 1291.4 (M−H)⁻.

2.57.24-[(1E)-3-({[2-({3-[(4-{2-carboxy-6-[8-([1,3]thiazolo[4,5-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)prop-1-en-1-yl]-2-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-beta-alanyl}amino)phenylbeta-D-glucopyranosiduronic Acid

The title compound was prepared by substituting Example 1.57.1 forExample 1.2.9 in Example 2.1. ¹H NMR (500 MHz, dimethyl sulfoxide-d₆) δppm 9.03 (s, 1H), 8.72 (d, 1H), 8.66 (d, 1H), 8.25 (s, 1H), 7.89 (br m,1H), 7.65 (d, 1H), 7.52 (br m, 2H), 7.46 (d, 1H), 7.39 (t, 1H), 7.30 (s,1H), 7.11 (br d, 1H), 7.03 (d, 1H), 6.98 (s, 2H), 6.97 (d, 1H), 6.58 (m,1H), 6.15 (m, 1H), 4.96 (s, 2H), 4.88 (br m, 1H), 4.64 (br m, 2H), 3.88(m, 3H), 3.79 (br m, 2H), 3.27-3.48 (m, 14H), 3.01 (m, 2H), 2.67 (br m,2H), 2.54 (m, 2H), 2.09 (s, 3H), 2.03 (t, 2H), 1.45 (m, 6H), 1.37 (br m,2H), 1.28-0.90 (m, 10H), 0.77-0.82 (m, 6H). MS (ESI) m/e 1484.4 (M−H)⁻.

2.58 Synthesis of4-[(1E)-3-({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)prop-1-en-1-yl]-2-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-beta-alanyl}amino)phenylbeta-D-glucopyranosiduronic Acid (Synthon HD) 2.58.13-(1-((3-(2-(((((E)-3-(3-(3-aminopropanamido)-4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)phenyl)allyl)oxy)carbonyl)(2-sulfoethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid

The title compound was prepared by substituting Example 1.2.9 forExample 1.22.5 in Example 2.56.1. MS (ESI−) m/e 1290.2 (M−H)⁻.

2.58.24-[(1E)-3-({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)prop-1-en-1-yl]-2-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-beta-alanyl}amino)phenylbeta-D-glucopyranosiduronic Acid

The title compound was prepared by substituting Example 1.58.1 forExample 1.56.1 in Example 2.56.2. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 9.03 (s, 1H), 8.25 (s, 1H), 8.03 (d, 1H), 7.89 (brm, 1H), 7.79 (d, 1H), 7.61 (d, 1H), 7.53 (br In, 1H), 7.46 (m, 2H), 7.37(m, 2H), 7.32 (s, 1H), 7.11 (br d, 1H), 7.03 (d, 1H), 6.98 (s, 2H), 6.97(d, 1H), 6.58 (m, 1H), 6.15 (m, 1H), 4.96 (s, 2H), 4.88 (br m, 1H), 4.64(br m, 2H), 3.88 (m, 3H), 3.79 (br m, 2H), 3.27-3.48 (m, 14H), 3.01 (m,2H), 2.67 (br m, 2H), 2.54 (m, 2H), 2.09 (s, 3H), 2.03 (t, 2H), 1.45 (m,6H), 1.37 (br m, 2H), 1.28-0.90 (m, 10H), 0.77-0.82 (m, 6H). MS (ESI−)m/e 1483.3 (M−H)⁻.

2.59 Synthesis of4-[(1E)-3-({[2-({3-[(4-{2-carboxy-6-[8-([1,3]thiazolo[5,4-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](3-phosphonopropyl)carbamoyl}oxy)prop-1-en-1-yl]-2-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-beta-alanyl}amino)phenylbeta-D-glucopyranosiduronic Acid (Synthon HS) 2.59.13-(1-((3-(2-(((((E)-3-(3-(3-aminopropanamido)-4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)phenyl)allyl)oxy)carbonyl)(3-phosphonopropyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(thiazolo[5,4-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid

The title compound was prepared by substituting Example 1.40.2 forExample 1.22.5 in Example 2.56.1. MS (ESI−) m/e 1305.4 (M−H)⁻.

2.59.24-[(1E)-3-({[2-({3-[(4-{2-carboxy-6-[8-([1,3]thiazolo[5,4-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](3-phosphonopropyl)carbamoyl}oxy)prop-1-en-1-yl]-2-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-beta-alanyl}amino)phenylbeta-D-glucopyranosiduronic Acid

The title compound was prepared by substituting Example 1.59.1 forExample 1.56.1 in Example 2.56.2. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 9.03 (s, 1H), 8.53 (dd, 1H), 8.24 (s, 1H), 8.16 (dd,1H), 7.90 (br s, 1H), 7.61 (d, 1H), 7.54 (d, 1H) 7.52 (d, 1H), 7.44 (d,1H), 7.37 (t, 1H), 7.28 (s, 1H), 7.11 (br d, 1H), 7.03 (d, 1H), 6.98 (s,2H), 6.97 (d, 1H), 6.56 (m, 1H), 6.16 (m, 1H), 4.96 (s, 2H), 4.86 (br m,1H), 4.64 (br d, 2H), 3.88 (m, 3H), 3.79 (br m, 2H), 3.27-3.44 (m, 14H),3.01 (m, 2H), 2.54 (m, 2H), 2.08 (s, 3H), 2.03 (t, 2H), 1.46 (m, 6H),1.37 (br m, 2H), 1.28-0.90 (m, 10H), 0.77-0.82 (m, 6H). MS (ESI) m/e1498.4 (M−H)⁻.

2.60 Synthesis of4-[(1E)-3-({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-5-(3-phosphonopropoxy)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)-]dec-1-yl}oxy)ethyl](methyl)carbamoyl}oxy)prop-1-en-1-yl]-2-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-beta-alanyl}amino)phenylbeta-D-glucopyranosiduronic Acid (Synthon HW) 2.60.13-(1-(((3-(2-(((((E)-3-(3-(3-aminopropanamido)-4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)phenyl)allyl)oxy)carbonyl)(methyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-5-(3-phosphonopropoxy)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid

The title compound was prepared by substituting Example 1.31.11 forExample 1.22.5 in Example 2.56.1. MS (ESI) m/e 1336.2 (M+Na)⁺.

2.60.24-[(1E)-3-({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-5-(3-phosphonopropoxy)-3,4-dihydroisoquinolin-2(H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](methyl)carbamoyl}oxy)prop-1-en-1-yl]-2-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-beta-alanyl}amino)phenylbeta-D-glucopyranosiduronic Acid

The title compound was prepared by substituting Example 1.60.1 forExample 1.56.1 in Example 2.56.2. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 9.03 (s, 1H) 8.25 (s, 1H), 8.01 (d, 1H), 7.83-7.91(m, 1H), 7.75 (dd, 2H), 7.42-7.58 (m, 2H), 7.34 (t, 1H), 7.28 (s, 1H),6.93-7.15 (m, 6H), 6.56 (d, 1H), 6.09-6.24 (m, 1H), 5.01 (s, 3H),4.80-4.92 (m, 2H), 4.57-4.69 (m, 3H), 4.12-4.21 (m, 6H), 3.86-3.94 (m,7H), 3.28-3.47 (m, 12H), 2.77-2.96 (m, 6H), 2.52-2.58 (m, 2H), 2.09 (s,3H), 1.90-2.05 (m, 4H), 1.65-1.78 (m, 2H), 0.90-1.53 (m, 16H), 0.80 (m,6H). MS (ESI) m/e 1529.5 (M+H)⁺.

2.61 Synthesis of4-[(1E)-3-([2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1j]dec-1-yl}oxy)ethyl](3-phosphonopropyl)carbamoyl)oxy)prop-1-en-1-yl]-2-((N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-beta-alanyl)amino)phenylbeta-D-glucopyranosiduronic Acid (Synthon HX) 2.61.13-(1-((3-(2-(((((E)-3-(3-(3-aminopropanamido)-4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)phenyl)allyl)oxy)carbonyl)(3-phosphonopropyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid

The title compound was prepared by substituting Example 1.14.4 forExample 1.22.5 in Example 2.56.1. MS (ESI) m/e 1304.3 (M−H)⁻.

2.61.24-[(1E)-3-({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](3-phosphonopropyl)carbamoyl}oxy)prop-1-en-1-yl]-2-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-beta-alanyl}amino)phenylbeta-D-glucopyranosiduronic Acid

The title compound was prepared by substituting Example 1.61.1 forExample 1.56.1 in Example 2.56.2. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 9.03 (s, 1H), 8.25 (br s, 1H), 8.03 (d, 1H), 7.89(br m, 1H), 7.79 (d, 1H), 7.61 (d, 1H), 7.53 (br m, 1H), 7.46 (m, 2H),7.37 (m, 2H), 7.28 (s, 1H), 7.11 (br d, 1H), 7.03 (d, 1H), 6.98 (s, 2H),6.97 (d, 1H), 6.56 (m, 1H), 6.17 (m, 1H), 4.96 (s, 2H), 4.86 (br m, 1H),4.64 (br d, 2H), 3.88 (m, 3H), 3.79 (br m, 2H), 3.27-3.44 (m, 14H), 3.01(m, 2H), 2.54 (m, 2H), 2.08 (s, 3H), 2.03 (t, 2H), 1.46 (m, 6H), 1.37(br m, 2H), 1.28-0.90 (m, 10H), 0.77-0.82 (m, 6H). MS (ESI−) m/e 1497.4(M−H)⁻.

2.62 Synthesis of4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-3-[2-(2-{[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]amino}ethoxy)ethoxy]phenylbeta-D-glucopyranosiduronic Acid (Synthon HY) 2.62.1(2S,3R,4S,5S,6S)-2-(4-formyl-3-hydroxyphenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate

2,4-Dihydroxybenzaldehyde (15 g) and(2S,3R,4S,5S,6S)-2-bromo-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (10 g) were dissolved in acetonitrile followed by theaddition of silver carbonate (10 g) and the reaction was heated to 49°C. After stirring for 4 hours, the reaction was cooled, filtered andconcentrated. The crude title compound was suspended in dichloromethaneand was filtered through diatomaceous earth and concentrated. Theresidue was purified by silica gel chromatography eluting with 1-100%ethyl acetate/heptane to provide the title compound.

2.62.2(2S,3R,4S,5S,6S)-2-(3-hydroxy-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate

A solution of Example 2.62.1 (16.12 g) in tetrahydrofuran (200 mL) andmethanol (200 mL) was cooled to 0° C., and sodium borohydride (1.476 g)was added portionwise. The reaction was stirred for 20 minutes and wasquenched with a 1:1 mixture of water:aqueous saturated sodiumbicarbonate solution (400 mL). The resulting solids were filtered offand rinsed with ethyl acetate. The phases were separated and the aqueouslayer was extracted four times with ethyl acetate. The combined organiclayers were dried over magnesium sulfate, filtered, and concentrated.The crude title compound was purified via silica gel chromatographyeluting with 1-100% ethyl acetate/heptanes to provide the titlecompound. MS (ESI) m/e 473.9 (M+NH₄)⁺.

2.62.3(2S,3R,4S,5S,6S)-2-(4-(((tert-butyldimethylsilyl)oxy)methyl)-3-hydroxyphenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate

Example 2.62.2 (7.66 g) and tert-butyldimethylsilyl chloride (2.78 g) indichloromethane (168 mL) at −5° C. was added imidazole (2.63 g) and thereaction was stirred overnight allowing the internal temperature of thereaction to warm to 12° C. The reaction mixture was poured intosaturated aqueous ammonium chloride and extracted four times withdichloromethane. The combined organics were washed with brine, driedover magnesium sulfate, filtered and concentrated. The crude titlecompound was purified via silica gel chromatography eluting with 1-50%ethyl acetate/heptanes to provide the title compound. MS (ESI) m/e 593.0(M+Na)⁺.

2.62.4(2S,3R,4S,5S,6S)-2-(3-(2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethoxy)ethoxy)-4-(((tert-butyldimethylsilyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate

To Example 2.62.3 (5.03 g) and triphenylphosphine (4.62 g) in toluene(88 mL) was added di-tert-butyl-azodicarboxylate (4.06 g) and thereaction was stirred for 30 minutes. (9H-Fluoren-9-yl)methyl(2-(2-hydroxyethoxy)ethyl)carbamate was added and the reaction wasstirred for an addition 1.5 hours. The reaction was loaded directly ontosilica gel and was eluted with 1-50% ethyl acetate/heptanes to providethe title compound.

2.62.5(2S,3R,4S,5S,6S)-2-(3-(2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethoxy)ethoxy)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate

Example 2.62.4 (4.29 g) was stirred in a 3:1:1 solution of aceticacid:water:tetrahydrofuran (100 mL) overnight. The reaction was pouredinto saturated aqueous sodium bicarbonate and extracted with ethylacetate. The organic layer was dried over magnesium sulfate, filteredand concentrated. The crude title compound was purified via silica gelchromatography, eluting with 1-50% ethyl acetate/heptanes to provide thetitle compound.

2.62.6(2S,3R,4S,5S,6S)-2-(3-(2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethoxy)ethoxy)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate

To a solution of Example 2.62.5 (0.595 g) and bis(4-nitrophenyl)carbonate (0.492 g) in N,N-dimethylfornamide (4 mL) was addedN-ethyl-N-isopropylpropan-2-amine (0.212 mL). After 1.5 hours, thereaction was concentrated under high vacuum. The reaction was loadeddirectly onto silica gel and eluted using 1-50% ethyl acetate/heptanesto provide the title compound. MS (ESI) m/e 922.9 (M+Na)⁺.

2.62.73-(1-((3-(2-((((2-(2-(2-aminoethoxy)ethoxy)-4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)(2-sulfoethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid

To a solution of Example 1.2.9 (0.073 g) and Example 2.62.6 (0.077 g) inN,N-dimethylformamide (0.5 mL) was added N,N-diisopropylethylamine(0.066 mL), and the reaction was stirred overnight. The reaction wasconcentrated, and the residue was dissolved in tetrahydrofuran (0.5 mL)and methanol (0.5 mL) and treated with lithium hydroxide monohydrate(0.047 g) as a solution in water (0.5 mL). After 1 hour, the reactionwas diluted with N,N-dimethylformamide and water and was quenched by theaddition of trifluoroacetic acid (0.116 mL). The mixture was purified byreverse phase HPLC using a Gilson system, eluting with 10-75%acetonitrile in water containing 0.1% v/v trifluoroacetic acid. Thedesired fractions were combined and freeze-dried to provide the titlecompound.

2.62.84-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-3-[2-(2-{[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]amino}ethoxy)ethoxy]phenylbeta-D-glucopyranosiduronic Acid

A solution of Example 2.62.7 (0.053 g), 2,5-dioxopyrrolidin-1-yl3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate (0.012 g) andN,N-diisopropylethylamine (0.033 mL) in N,N-dimethylformamide (0.75 mL)was stirred at room temperature. After stirring for 1 hour, the reactionwas diluted with N,N-dimethylformamide and water. The mixture waspurified by reverse phase HPLC using a Gilson system, eluting with10-75% acetonitrile in water containing 0.1% v/v trifluoroacetic acid.The desired fractions were combined and freeze-dried to provide thetitle compound. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm 12.85 (s,1H), 8.04 (d, 2H), 7.79 (d. 1H), 7.61 (d, 1H), 7.54 (d, 1H), 7.51-7.40(m, 2H), 7.40-7.31 (m, 3H), 7.20 (d, 1H), 7.00-6.94 (m, 3H), 6.73-6.57(m, 2H), 5.06 (t, 1H), 5.01-4.91 (m, 4H), 3.96-3.85 (m, 2H), 3.85-3.78(m, 2H), 3.78-3.69 (m, 2H), 3.59 (t, 2H), 3.53-3.34 (m, 6H), 3.34-3.21(m, 4H), 3.17 (q, 2H), 3.02 (t, 2H), 2.66 (t, 2H), 2.33 (t, 2H), 2.10(s, 3H), 1.44-0.90 (m, 16H), 0.83 (d, 6H). MS (−ESI) m/e 1432.4 (M−H)⁻.

2.63 Synthesis of4-[(1E)-3-({[2-({(3-[(4-{2-carboxy-6-[8-([1,3]thiazolo[4,5-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](3-phosphonopropyl)carbamoyl}oxy)prop-1-en-1-yl]-2-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-beta-alanyl}amino)phenylbeta-D-glucopyranosiduronic Acid (Synthon IB) 2.63.13-(1-((3-(2-(((((E)-3-(3-(3-aminopropanamido)-4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)phenyl)allyl)oxy)carbonyl)(3-phosphonopropyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(thiazolo[4,5-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid

The title compound was prepared by substituting Example 1.39.2 forExample 1.22.5 in Example 2.56.1.

2.63.24-[(1E)-3-({[2-({3-[(4-{2-carboxy-6-[8-([1,3]thiazolo[4,5-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](3-phosphonopropyl)carbamoyl}oxy)prop-1-en-1-yl]-2-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-beta-alanyl}amino)phenylbeta-D-glucopyranosiduronic Acid

The title compound was prepared by substituting Example 2.63.1 forExample 1.56.1 in Example 2.56.2. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 9.03 (s, 1H), 8.61 (d, 1H), 8.55 (d, 1H), 8.25 (brs, 1H), 7.89 (br m, 1H), 7.65 (d, 1H), 7.50 (br d, 1H), 7.46 (d, 1H),7.39 (m, 2H), 7.28 (s, 1H), 7.11 (br d, 1H), 7.03 (d, 1H), 6.98 (s, 2H),6.97 (d, 1H), 6.56 (m, 1H), 6.17 (m, 1H), 4.97 (s. 2H), 4.86 (br m, 1H),4.64 (br d, 2H), 3.88 (m, 3H), 3.79 (br m, 2H), 3.27-3.44 (m, 14H), 3.01(m, 2H), 2.54 (m, 2H), 2.08 (s, 3H), 2.03 (t, 2H), 1.46 (m, 6H), 1.37(br m, 2H), 1.28-0.90 (m, 10H), 0.77-0.82 (m, 6H). MS (ESI) m/e 1498.3(M−H)⁻.

2.64 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[(2-carboxyethyl)({[(2E)-3-(4-{[(2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl]oxy}-3-[(3-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}propanoyl)amino]phenyl)prop-2-en-1-yl]oxy}carbonyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicacid (Synthon IE) 2.64.13-(1-((3-(2-(((((E)-3-(3-(3-aminopropanamido)-4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)phenyl)allyl)oxy)carbonyl)(2-carboxyethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid, Trifluoroacetic Acid Salt

To a solution of Example 1.25.2 (0.050 g) and Example 2.44.7 (0.061 g)in N,N-dimethylformamide (1 mL) was added N,N-diisopropylethylamine(0.047 mL), and the reaction was stirred at room temperature overnight.The reaction was concentrated, and the residue was dissolved in methanol(0.5 mL) and tetrahydrofuran (0.5 mL) and treated with a solution oflithium hydroxide hydrate (0.034 g) in water (0.5 mL). The reaction wasstirred at room temperature for 1 hour. The reaction was quenched withtrifluoroacetic acid (0.083 mL) and diluted with N,N-dimethylformamide(1 mL). The mixture was purified by reverse phase HPLC using a Gilsonsystem, eluting with 10-75% acetonitrile in water containing 0.1% v/vtrifluoroacetic acid. The desired fractions were combined andfreeze-dried to provide the title compound

2.64.26-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[(2-carboxyethyl)({[(2E)-3-(4-{[(2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl]oxy}-3-[(3-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}propanoyl)amino]phenyl)prop-2-en-1-yl]oxy}carbonyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid

To a solution of Example 2.64.1 (0.042 g) and2,5-dioxopyrrolidin-1-yl-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate(10 mg) in N,N-dimethylformamide (0.5 mL) was addedN,N-diisopropylethylamine (0.027 mL), and the reaction was stirred atroom temperature for 2 hours. The reaction was diluted withN,N-dimethylformamide (1 mL) and water (0.5 mL). The mixture waspurified by reverse phase HPLC using a Gilson system, eluting with10-75% acetonitrile in water containing 0.1% v/v trifluoroacetic acid.The desired fractions were combined and freeze-dried to provide thetitle compound. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm 12.85 (s,1H), 9.04 (s, 1H), 8.25 (s, 1H), 8.03 (d, 1H), 7.87 (t, 1H), 7.79 (d,1H), 7.61 (d, 1H), 7.54-7.40 (m, 3H), 7.40-7.31 (m, 2H), 7.28 (s, 1H),7.10 (d, 1H), 7.04 (d, 1H), 6.98 (s, 2H), 6.95 (d, 1H), 6.57 (d, 1H),6.24-6.11 (m, 1H), 4.96 (s, 2H), 4.86 (t, 1H), 4.65 (d, 2H), 3.95-3.84(m 2H), 3.84-3.75 (m, 4H), 3.44-3.24 (m, 10H), 3.01 (t, 2H), 2.62-2.52(m, 4H), 2.09 (s, 3H), 2.03 (t, 2H), 1.46 (h, 4H), 1.40-1.31 (m, 2H),1.30-0.88 (m, 14H), 0.87-0.75 (m, 6H). MS (ESI) m/e 1447.5 (M−H)⁻.

2.65 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[(2-carboxyethyl){[(4-{[(2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl]oxy}-2-[2-(2-{[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]amino}ethoxy)ethoxy]benzyl)oxy]carbonyl}amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (Synthon 1) 2.65.13-(1-((3-(2-((((2-(2-(2-aminoethoxy)ethoxy)-4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)(2-carboxyethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid

A solution of Example 1.25.2 (0.055 g,). Example 2.62.6 (0.060 g) andN,N-diisopropylethylamine (0.052 mL) in N,N-dimethylformamide (0.4 mL)as stirred overnight. The reaction was concentrated, and the residue wasdissolved in tetrahydrofuan (0.5 mL), methanol (0.5 mL) then treatedwith lithium hydroxide hydrate (0.037 g) as a solution in water (0.5mL). After stirring for 1 hour, the reaction was quenched withtrifluoroacetic acid (0.091 mL) and diluted with N,N-dimethylformamide(1 mL). The mixture was purified by reverse phase HPLC using a Gilsonsystem, eluting with 10-75% acetonitrile in water containing 0.1% v/vtrifluoroacetic acid. The desired fractions were combined andfreeze-dried to provide the title compound as the trifluoroacetic acidsalt.

2.65.26-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[(2-carboxyethyl){[(4-([(2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl]oxy}-2-[2-(2-{[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]amino}ethoxy)ethoxy]benzyl)oxy]carbonyl}amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylicAcid

A solution of the trifluoroacetic acid salt of Example 2.65.1 (0.043).2,5-dioxopyrrolidin-1-yl3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate (10 mg) andN,N-diisopropylethylamine (0.028 mL) were stirred together inN,N-dimethylformamide (1 mL) at room temperature. After stirring for 1hour, the reaction was diluted with N,N-dimethylformamide (0.5 mL) andwater (0.5 mL). The mixture was purified by reverse phase HPLC using aGilson system, eluting with 5-75% acetonitrile in water containing 0.1%v/v trifluoroacetic acid. The desired fractions were combined andfreeze-dried to provide the title compound. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 12.84 (s, 1H), 8.03 (d, 1H), 8.00 (t, 1H), 7.79 (d,1H), 7.62 (d, 1H), 7.54-7.41 (m, 3H), 7.36 (td, 2H), 7.29 (s, 1H), 7.19(d, 1H), 6.97 (s, 2H), 6.95 (d, 1H), 6.67 (d, 1H), 6.60 (dd, 1H),5.14-5.03 (m, 1H), 4.96 (d, 4H), 4.08 (tt, 4H), 3.89 (q, 4H), 3.84-3.77(m, 2H), 3.71 (t, 2H), 3.59 (t, 2H), 3.52-3.35 (m, 6H), 3.28 (dq. 4H),3.17 (q, 2H), 3.01 (t, 2H), 2.46 (d, 1H), 2.33 (t, 2H), 2.09 (s, 3H),1.45-0.90 (m. 12H), 0.82 (d, 6H). MS (ESI) m/e 1396.4 (M−H)⁻.

2.66 Synthesis ofN-[6-(ethenylsulfonyl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide(Synthon KY) 2.66.13-(1-((3-(2-((((4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)(2-sulfoethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid

To a mixture of Example 1.2.9 (57 mg) and (9H-fluoren-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-1-oxobutan-2-yl)carbamate(54 mg) in N,N-dimethylformamide (2 mL) was addedN,N-diisopropylethylamine (103 μL). The mixture was stirred overnight,and diethylamine (61.5 μL) was added. The resulting mixture was stirredfor 4 hours and purified by reverse phase HPLC using a Gilson system andC18 column, eluting with 10-70% acetonitrile in water containing 0.1%v/v trifluoroacetic acid, to provide the title compound. MS (ESI) m/e1257.4 (M−H).

2.66.2N-[6-(ethenylsulfonyl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide

The title compound was prepared using the procedure in Example 2.83,replacing Example 1.2.9 and 2,5-dioxopyrrolidin-1-yl6-(2-chloroacetamido)hexanoate with Example 2.66.1 and Example 2.82.5,respectively. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm 12.88 (s,0H), 9.99 (s, 1H), 8.05 (t, 2H), 7.80 (t, 2H), 7.60 (q, 3H), 7.36 (td,2H), 7.28 (d, 3H), 7.01-6.89 (m, 2H), 6.29-6.15 (m, 2H), 6.02 (s, 1H),4.97 (d, 4H), 4.40 (td, 1H), 4.20 (t, 1H), 4.00-3.77 (m, 4H), 3.55-3.33(m, 4H), 3.25 (d, 2H), 3.14-2.88 (m, 6H), 2.62 (t, 2H), 2.09 (s, 4H),1.82-0.90 (m, 10H), 0.84 (dd, 13H). MS (ESI) m/e 1447.2 (M+H).

2.67 Synthesis of4-[(1E)-3-{[(4-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](3-phosphonopropyl)amino}piperidin-1-yl)carbonyl]oxy}prop-1-en-1-yl]-2-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-beta-alanyl}amino)phenylbeta-D-glucopyranosiduronic Acid (Synthon IW) 2.67.13-(1-((3-(2-((1-((((E)-3-(3-(3-aminopropanamido)-4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)phenyl)allyl)oxy)carbonyl)piperidin-4-yl)(3-phosphonopropyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid

To a solution of Example 1.26.2 (0.045 g) and Example 2.44.7 (0.053 g)in N,N-dimethylformamide (1 mL) was added N,N-diisopropylethylamine(0.041 mL), and the reaction was stirred at room temperature overnight.The reaction was concentrated, and the residue was dissolved in methanol(0.5 mL) and tetrahydrofuran (0.5 mL) and treated with a solution oflithium hydroxide monohydrate (0.030 g) in water (0.5 mL) at roomtemperature. After stirring for 1 hour, the reaction was quenched withtrifluoroacetic acid (0.073 mL) and diluted with N,N-dimethylformamide(1 mL). The mixture was purified by reverse phase HPLC using a Gilsonsystem, eluting with 10-60% acetonitrile in water containing 0.1% v/vtrifluoroacetic acid. The desired fractions were combined andfreeze-dried to provide the title compound.

2.67.24-[(1E)-3-{([(4-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](3-phosphonopropyl)amino}piperidin-1-yl)carbonyl]oxy}prop-1-en-1-yl]-2-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-beta-alanyl}amino)phenylbeta-D-glucopyranosiduronic Acid

To a solution of Example 2.67.1 (0.040 g) and 2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (9.84 mg) inN,N-dimethylformamide (1 mL) was added N,N-diisopropylethylamine (0.023mL), and the reaction was stirred at room temperature for 2 hours. Thereaction was diluted with N,N-dimethylformamide (1 mL) and water (1 mL).The mixture was purified by reverse phase HPLC using a Gilson system,eluting with 10-60% acetonitrile in water containing 0.1% v/vtrifluoroacetic acid. The desired fractions were combined andfreeze-dried to provide the title compound. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 9.28 (s, 1H), 9.04 (s, 1H), 8.25 (s, 1H), 8.03 (d,1H), 7.87 (t, 1H), 7.79 (d, 1H), 7.62 (dd, 1H), 7.55-7.40 (m, 3H), 7.36(td. 2H), 7.29 (s, 1H), 7.11 (dd, 1H), 7.05 (d, 1H), 6.98 (s, 2H), 6.95(d, 1H), 6.59 (d, 1H), 6.20 (t, 1H), 6.16 (t, OH), 4.96 (s, 2H), 4.88(d, 1H), 4.66 (d, 2H), 4.14 (d, 2H), 3.96-3.86 (m, 2H), 3.83 (s, 2H),3.54 (t, 7H), 3.48-3.28 (m, 12H), 3.01 (t, 2H), 2.84 (s, 2H), 2.55 (t,2H), 2.10 (s, 3H), 2.07-1.95 (m. 4H), 1.88 (s, 2H), 1.73-1.54 (m, 4H),1.54-1.38 (m 6H), 1.39-1.26 (m, 4H), 1.26-0.93 (m, 8H), 0.86 (s, 6H). MS(ESI) m/e 1582.4 (M+H)⁺.

2.68 Synthesis of4-[(1E)-3-{[(4-{[2-({3-[(4-{2-carboxy-6-[8-([1,3]thiazolo[4,5-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](3-phosphonopropyl)amino}piperidin-1-yl)carbonyl]oxy}prop-1-en-1-yl]-2-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-beta-alanyl}amino)phenylbeta-D-glucopyranosiduronic Acid (Synthon IY) 2.68.13-(1-((3-(2-((1-((((E)-3-(3-(3-aminopropanamido)-4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)phenyl)allyl)oxy)carbonyl)piperidin-4-yl)(3-phosphonopropyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(thiazolo[4,5-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid

The title compound was prepared by substituting Example 1.50.2 forExample 1.44.7 in Example 2.56.1. MS (ESI) m/e 1388.5 (M−H)⁻.

2.68.24-[(1E)-3-{[(4-{[2-({3-[(4-{2-carboxy-6-[8-([1,3]thiazolo[4,5-b]pyridin-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]pyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](3-phosphonopropyl)amino}piperidin-1-yl)carbonyl]oxy}prop-1-en-1-yl]-2-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-beta-alanyl}amino)phenylbeta-D-glucopyranosiduronic Acid

The title compound was prepared by substituting Example 1.68.1 forExample 1.56.1 in Example 2.56.2. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 9.03 (s, 1H), 8.61 (d, 1H), 8.50 (d, 1H), 8.25 (brs, 1H), 7.89 (t, 1H), 7.65 (d, 1H), 7.49 (d, 1H), 7.46 (d, 1H), 7.36 (m,2H), 7.29 (s, 1H), 7.11 (br d, 1H), 7.03 (d, 1H), 6.98 (s, 2H), 6.97 (d,1H), 6.58 (m, 1H), 6.17 (m, 1H), 4.97 (s, 2H), 4.88 (d, 1H), 4.65 (br d,2H), 3.88 (m, 3H), 3.79 (br m, 2H), 3.66 (br m, 2H), 3.27-3.44. (m,14H), 3.01 (m, 2H), 2.85 (br m, 2H), 2.54 (m, 2H), 2.10 (s, 3H), 2.03(t, 2H), 1.98 (br m, 2H), 1.89 (m, 1H), 1.62 (m, 4H), 1.46 (m, 6H), 1.31(m, 4H), 1.15 (m, 6H), 1.04 (m, 2H), 0.86 (s, 6H). MS (ESI) m/c 1581.4(M−H)⁻.

2.69 Synthesis of4-[(1E)-3-({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)prop-1-en-1-yl]-2-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-beta-alanyl}amino)phenylbeta-D-glucopyranosiduronic Acid (Synthon JA) 2.69.13-(1-((3-(2-(((((E)-3-(3-(3-aminopropanamido)-4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)phenyl)allyl)oxy)carbonyl)(2-sulfoethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)naphthalen-2-yl)picolinicAcid

The title compound was prepared by substituting Example 1.43.7 forExample 2.44.7 in Example 2.56.1. MS (ESI) m/e 1309.1 (M+Na)⁺.

2.69.24-[(1E)-3-({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)prop-1-en-1-yl]-2-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-beta-alanyl}amino)phenylbeta-D-glucopyranosiduronic Acid

The title compound was prepared by substituting Example 2.69.1 forExample 2.56.1 in Example 2.56.2. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 13.09 (s, 1H), 9.02 (s, 2H), 8.35 (d, 1H), 8.13-8.29(m, 4H), 7.86-8.09 (m, 5H), 7.81 (d, 1H), 7.66-7.75 (m, 1H), 7.44-7.55(m, 1H), 7.37 (t, 1H), 7.09-7.18 (m, 1H), 7.03 (d, 1H), 6.98 (s, 1H),6.48-6.62 (m, 1H), 6.07-6.22 (m, 1H), 4.81-4.92 (m, 1H), 4.58-4.74 (m,2H), 3.80-3.93 (m, 3H), 3.27-3.37 (m, 5H), 2.53-2.68 (m, 4H), 2.15-2.23(m, 3H), 2.03 (t, 2H), 1.36-1.53 (m, 6H), 0.97-1.33 (m, 24H), 0.81 (d,6H). MS (ESI) m/c 1478.3 (M−H)⁻.

2.70 This Paragraph was Intentionally Left Blank 2.71 This Paragraph wasIntentionally Left Blank 2.72 This Paragraph was Intentionally LeftBlank 2.73 This Paragraph was Intentionally Left Blank 2.74 ThisParagraph was Intentionally Left Blank 2.75 This Paragraph wasIntentionally Left Blank 2.76 This Paragraph was Intentionally LeftBlank 2.77 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-[1-({3-[2-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-3-sulfo-L-alanyl}amino)ethoxy]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylicAcid (Synthon FA)

To a solution of Example 1.15 (0.023 g) and 2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (9.12 mg) inN,N-dimethylformamide (0.5 mL) was added N,N-diisopropylethylamine(0.012 mL), and the reaction was stirred overnight. The reaction wasdiluted with N,N-dimethylformamide (1 mL) and water (0.5 mL). Themixture was purified by reverse phase HPLC using a Gilson system,eluting with 10-85% acetonitrile in water containing 0.1% v/vtrifluoroacetic acid. The desired fractions were combined andfreeze-dried to provide the title compound. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 12.84 (s, 1H), 8.04 (d, 1H), 7.90 (d, 1H), 7.79 (d,1H), 7.65-7.57 (m, 2H), 7.54 (d, 1H), 7.51-7.41 (m, 2H), 7.40-7.31 (m,3H), 7.01-6.96 (m, 3H), 4.96 (s, 2H), 4.34-4.28 (m, 3H), 3.89 (t, 2H),3.83 (s, 2H), 3.37 (t, 2H), 3.29 (t, 2H), 3.16-2.95 (m, 4H), 2.80 (dd,1H), 2.70 (dd, 1H), 2.11 (s, 3H), 2.06 (t, 2H), 1.47 (tt, 4H), 1.40-0.92(m, 12H), 0.84 (s, 6H). MS (ESI) m/e 1090.3 (M+H)⁺.

2.78 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-[1-(3-[2-(2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl](2-sulfoethyl)amino)ethoxy)ethoxy]-5,7-dimethyltricyclo[3.3.1.1¹⁷]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylicAcid (Synthon FJ)

The title compound was prepared as described in Example 2.1, replacingExample 1.2.9 and4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl) carbonate with Example 1.11.4 and perfluorophenyl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate, respectively. ¹H NMR(400 MHz, dimethyl sulfoxide-d₆) δ ppm 12.84 (s, 1H), 8.04 (d, 1H), 7.79(d, 1H), 7.61 (d, 1H), 7.52 (dd, 1H), 7.42-7.49 (m, 2H), 7.33-7.39 (m,2H), 7.30 (s, 1H), 6.98 (s, 2H), 6.96 (d, 1H), 4.95 (s, 2H), 3.89 (t,2H), 3.82 (s, 2H), 3.46-3.56 (m, 4H), 3.31-3.46 (m, 10H), 3.01 (t, 2H),2.61-2.68 (m, 1H), 2.55-2.60 (m, 1H), 2.21-2.32 (m, 2H), 2.10 (s, 3H),1.40-1.51 (m, 4H), 1.37 (d, 2H), 0.91-1.30 (m, 12H), 0.83 (s, 6H). MS(ESI) m/e 1091.2 (M+H)⁺.

2.79 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3-(2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl](2-sulfoethyl)amino}ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylicAcid (Synthon FK)

The title compound was prepared as described in Example 2.1, replacing4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl) carbonate with perfluorophenyl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate. ¹H NMR (400 MHz,dimethyl sulfoxide-d₆) δ ppm 12.85 (s, 1H), 8.04 (d, 1H), 7.79 (d, 1H),7.61 (d, 1H), 7.52 (dd, 1H), 7.41-7.49 (m, 2H), 7.32-7.39 (m, 2H), 7.28(s, 1H), 6.93-6.98 (m, 3H), 4.95 (s, 2H), 3.89 (t, 2H), 3.81 (s, 2H),3.32-3.38 (m, 2H), 3.21-3.27 (m, 2H), 3.01 (t, 2H), 2.61-2.67 (m, 2H),2.53-2.58 (m, 2H), 2.33-2.39 (m, 1H), 2.20-2.29 (m, 2H), 2.09 (s, 3H),1.40-1.51 (m, 4H), 1.34 (s, 2H), 0.93-1.27 (m, 13H), 0.83 (s, 6H). MS(ESI) m/e 1047.2 (M+H)⁺.

2.80 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo-22-(2-sulfoethyl)-3,6,9,12,15,18-hexaoxa-22-azatetracosan-24-yl]oxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (Synthon FQ)

The title compound was prepared as described in Example 2.1, replacing4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl) carbonate with perfluorophenyl1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,9,12,15,18-pentaoxahenicosan-2-oate.¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm 12.84 (s, 1H), 8.04 (d,1H), 7.79 (d, 1H), 7.61 (d, 1H), 7.42-7.54 (m, 3H), 7.33-7.38 (m, 2H),7.28 (s, 1H), 6.95 (dd, 1H), 4.95 (s, 2H), 3.89 (t, 2H), 3.81 (s, 2H),3.07-3.53 (m, 24H), 3.01 (t, 2H), 2.61-2.69 (m, 1H), 2.54-2.60 (m, 1H),2.09 (s, 3H), 1.96 (d, 2H), 0.92-1.39 (m, 13H), 0.84 (s. 6H). MS (ESI)m/e 1269.4 (M+H)⁺.

2.81 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo-22-(2-sulfoethyl)-3,6,9,12,15,18,25-heptaoxa-22-azaheptacosan-27-yl]oxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)-]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (Synthon FR)

The title compound was prepared as described in Example 2.1, replacingExample 1.2.9 and4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl) carbonate with Example 1.11.4 and perfluorophenyl1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-oate,respectively. ¹H NMR (500 MHz, dimethyl sulfoxide-d₆) δ ppm 12.84 (s,1H), 8.04 (d, 1H), 7.79 (d, 1H), 7.61 (d, 1H), 7.52 (d, 1H), 7.41-7.50(m, 2H), 7.33-7.39 (m, 2H), 7.31 (s, 1H), 7.01 (d, 2H), 6.97 (d, 1H),4.96 (s, 2H), 3.89 (t, 2H), 3.83 (s, 2H), 3.31-3.60 (m, 30H), 3.01 (t,2H), 2.64-2.71 (m, 1H), 2.53-2.61 (m, 3H), 2.10 (s, 3H), 1.38 (s, 2H),1.20-1.31 (m, 4H), 1.12-1.18 (m, 2H), 0.91-1.12 (m, 4H), 0.84 (s, 6H).

2.82 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3-(2-{[6-(ethenylsulfonyl)hexanoyl](2-sulfoethyl)amino}ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylicAcid (Synthon JE) 2.82.1 Ethyl 6-((2-hydroxyethyl)thio)hexanoate

A mixture of ethyl 6-bromohexanoate (3 g), 2-mercaptoethanol (0.947 mL)and K₂CO₃ (12 g) in ethanol (100 mL) was stirred overnight and filtered.The filtrate was concentrated. The residue was dissolved indichloromethane (100 mL) and washed with water and brine. The organiclayer was dried over sodium sulfate, filtered, and concentrated toprovide the title compound.

2.82.2 6-((2-hydroxyethyl)thio)hexanoic Acid

A mixture of Example 2.82.1 (12 g) and 3 M aqueous NaOH solution (30 mL)in ethanol (30 mL) was stirred overnight. The organics were removedunder reduced pressure. The residual aqueous phase was washed with ethylacetate, acidified with HCl to pH 5 and extracted with dichloromethane.The extracts were combined, dried over sodium sulfate, filtered andconcentrated to provide the title compound.

2.82.3 6-((2-hydroxyethyl)sulfonyl)hexanoic Acid

To a stirred solution of Example 2.82.2 (4 g) in a mixture of water (40mL) and 1,4-dioxane (160 mL) was added Oxone (38.4 g), and the mixturewas stirred overnight. The mixture was filtered, and the filtrate wasconcentrated. The residual aqueous layer was extracted withdichloromethane. The extracts were combined and dried over sodiumsulfate, filtered, and concentrated to provide the title compound.

2.82.4 6-(vinylsulfonyl)hexanoic Acid

To a cold (0° C.) solution of Example 2.82.3 (1 g) in dichloromethane(10 mL) was added triethylamine (2.8 mL), followed by the addition ofmethanesulfonyl chloride (1.1 mL) under argon. The mixture was stirredovernight and washed with water and brine. The organic layer was driedover sodium sulfate, filtered, and concentrated to provide the titlecompound.

2.82.5 2,5-dioxopyrrolidin-1-yl-6-(vinylsulfonyl)hexanoate

To a stirred solution of Example 2.82.4 (0.88 g) in dichloromethane (10ml) was added 1-hydroxypyrrolidine-2,5-dione (0.54 g) andN,N′-methanediylidenedicyclohexanamine (0.92 g). The mixture was stirredovernight and filtered. The filtrate was concentrated and purified byflash chromatography, eluting with 10-25% ethyl acetate in petroleum, toprovide the title compound. MS (ESI) m/e 304.1 (M+1).

2.82.66-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3-(2-{[6-(ethenylsulfonyl)hexanoyl](2-sulfoethyl)amino}ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylicAcid

The title compound was prepared as described in Example 2.83, replacing2,5-dioxopyrrolidin-1-yl 6-(2-chloroacetamido)hexanoate with Example2.82.5. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm 12.86 (s, 1H),8.04 (d, 1H), 7.79 (d, 1H), 7.61 (d, 1H), 7.53 (dd, 1H), 7.42-7.49 (m,2H), 7.33-7.40 (m, 2H), 7.28 (s, 1H), 6.88-7.00 (m, 2H), 6.17-6.25 (m,2H), 4.95 (s. 2H), 3.89 (t, 2H), 3.81 (s, 2H), 3.38 (dd, 2H), 3.25 (t,2H), 3.04-3.12 (m, 2H), 3.01 (t, 2H), 2.62-2.69 (m, 1H), 2.56 (dd, 1H),2.27 (q, 2H), 2.09 (s, 3R), 1.53-1.62 (m, 2H), 1.43-1.51 (m, 2H),1.28-1.38 (m, 4H), 1.20-1.27 (m, 4H), 0.92-1.19 (m, 6H), 0.84 (s, 6H).MS (ESI) m/e 1042.2 (M+H)⁺.

2.83 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[{6-[(chloroacetyl)amino]hexanoyl}(2-sulfoethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (Synthon JM)

To a mixture of Example 1.2.9 (12.5 mg) and 2,5-dioxopyrrolidin-1-yl6-(2-chloroacetamido)hexanoate (6.7 mg) in N,N-dimethylfornamide (1.5mL) was added N,N-diisopropylethylamine (26 μL). The mixture was stirredfor 10 days and purified by reverse phase HPLC using a Gilson system andC18 column, eluting with 20-60% acetonitrile in water containing 0.1%v/v trifluoroacetic acid, to provide the title compound. ¹H NMR (500MHz, dimethyl sulfoxide-d₆) δ ppm 12.83 (s, 1H), 8.15-8.21 (m, 1H), 8.04(d, 1H), 7.79 (d, 1H), 7.61 (d, 1H), 7.52 (dd, 1H), 7.41-7.49 (m, 2H),7.32-7.39 (m, 2H), 7.28 (s, 1H), 6.96 (dd, 1H), 4.95 (s, 2H), 4.01 (d,2H), 3.89 (t, 2H), 3.81 (s, 2H), 3.39 (d, 2H), 3.25 (t, 2H), 2.98-3.10(m, 5H), 2.62-2.70 (m, 1H), 2.56-2.61 (m, 1H), 2.23-2.30 (m, 2H), 2.09(s, 3H), 1.33-1.52 (m, 5H), 1.19-1.30 (m, 6H), 0.91-1.18 (m, 6), 0.84(s, 6H). MS (ESI) m/e 1043.2 (M+H)⁺.

2.84 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](3-carboxypropyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide(Synthon LE)

A mixture of Example 1.56 (0.020 g),4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl) carbonate (0.022 g) and N,N-diisopropylethylamine (0.018mL) were stirred together in N,N-dimethylformamide (0.4 mL) at roomtemperature. After stirring for 5 hours, the reaction was diluted with a1:1 mixture of N,N-dimethylformamide and water (2 mL). The mixture waspurified by reverse phase HPLC using a Gilson system, eluting with10-85% acetonitrile in water containing 0.1% v/v trifluoroacetic acid.The desired fractions were combined and freeze-dried to provide thetitle compound. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm 12.82 (s,1H), 9.97 (s, 1H), 8.10-7.98 (m, 2H), 7.84-7.72 (m, 2H), 7.67-7.54 (m,3H), 7.54-7.41 (m, 3H), 7.40-7.32 (m, 2H), 7.30-7.23 (m, 3H), 6.99 (s,2H), 6.94 (d, 1H), 5.99 (s, 1H), 4.98 (s, 2H), 4.95 (s, 2H), 4.45-4.35(m, 2H), 4.19 (dd, 2H), 3.88 (t, 2H), 3.82-3.76 (m, 2H), 3.47-3.31 (m,4H), 3.28-3.19 (m, 4H), 3.07-2.89 (m, 4H), 2.21-2.11 (m, 4H), 2.0 (s,2H), 2.02-1.89 (m, 1H), 1.77-1.63 (m, 2H), 1.62-1.27 (m, 10H), 1.27-0.90(m, 13H), 0.88-0.78 (m, 12H); MS (ESI) m/e 1430.3 (M+1)⁺.

2.85 Synthesis ofN-{6-[(bromoacetyl)amino]hexanoyl}-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1.]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide(Synthon LH) 2.85.11H-benzo[d][1,2,3]triazol-1-yl6-(2-bromoacetamido)hexanoate

To a solution of 6-(2-bromoacetamido)hexanoic acid (105 mg) andbenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate(PyBOP. 325 mg) in N,N-dimethylformamide (3 mL) was added triethylamine(87 μL). The mixture was stirred for 1 hour and purified by a GilsonHPLC system (C18 column), eluting with 20-60% acetonitrile in 0.1% TFAwater to provide the title compound. MS (ESI) m/e 368.7 (M+H)⁺.

2.85.2N-{6-[(bromoacetyl)amino]hexanoyl}-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide

To a mixture of Example 2.6.1 (6.6 mg) and Example 2.85.2 (3.6 mg) inN,N-dimethylformamide (0.3 mL) was added N,N-diisopropylethylamine (2.52sL). The mixture was stirred for 5 minutes, diluted with dimethylsulfoxide and purified by reverse phase HPLC using a Gilson system andC18 column, eluting with 20-60% acetonitrile in water containing 0.1%v/v trifluoroacetic acid, to provide the title compound. ¹H NMR (500MHz, dimethyl sulfoxide-d₆) δ 6 9.99 (s, 1H), 8.24 (s, 1H), 8.08 (d,1H), 8.04 (d, 1H), 7.80 (dd, 2H), 7.60 (q, 3H), 7.56-7.50 (m, 1H),7.50-7.41 (m, 2H), 7.36 (q, 2H), 7.32-7.25 (m, 3H), 6.96 (d, 1H), 4.98(d, 4H), 4.39 (q, 1H), 4.20 (dd, 1H), 3.92-3.68 (m, 6H), 3.42 (dd, 1H),3.25 (t, 2H), 3.09-2.87 (m, 6H), 2.64 (s, 2H), 2.25-1.87 (m, 5H),1.79-0.89 (m, 17H), 0.88-0.67 (m, 12H). MS (ESI) m/e 1492.5 (M−H).

2.86 Synthesis of4-[([2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](3-carboxypropyl)carbamoyloxy)methyl]-3-[2-(2-{[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]amino}ethoxy)ethoxy]phenylbeta-D-glucopyranosiduronic Acid (Synthon IJ) 2.86.13-(1-((3-(2-((((2-(2-(2-aminoethoxy)ethoxy)-4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)(3-carboxypropyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid

To a solution of Example 1.56 (0.024 g) and Example 2.62.6 (0.030 g) inN,N-dimethylformamide (0.4 mL) was added N,N-diisopropylethylamine(0.025 mL), and the reaction was stirred overnight. The reaction wasconcentrated, and the residue dissolved in tetrahydrofuran (0.5 mL) andmethanol (0.5 mL) and treated with lithium hydroxide hydrate (0.018 g)as a solution in water (0.5 mL). After stirring for 1 hour, the reactionwas diluted with N,N-dimethylformamide (1 mL) and purified by reversephase HPLC using a Gilson system, eluting with 10-75% acetonitrile inwater containing 0.1% v/v trifluoroacetic acid. The desired fractionswere combined and freeze-dried to provide the title compound. MS (ESI)m/e 1262.7 (M+H)⁺.

2.86.24-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1l]dec-1-yl}oxy)ethyl](3-carboxypropyl)carbamoyl}oxy)methyl]-3-[2-(2-{[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]amino}ethoxy)ethoxy]phenylbeta-D-glucopyranosiduronic Acid

To a solution of Example 2.86.1 (0.0173 g) and 2,5-dioxopyrrolidin-1-yl3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate (4.38 mg) inN,N-dimethylformamide (0.8 mL) was added 2,5-dioxopyrrolidin-1-yl3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate (4.38 mg), and thereaction was stirred for 2 hours. The reaction was diluted with a 1:1mixture of N,N-dimethylformamide:water (1 mL), and the mixture waspurified by reverse phase HPLC using a Gilson system, eluting with10-80% acetonitrile in water containing 0.1% v/v trifluoroacetic acid.The desired fractions were combined and freeze-dried to provide thetitle compound. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm 12.77 (s,1H), 8.03 (d, 1H), 7.99 (t, 1H), 7.77 (d, 1H), 7.62 (d, 1H), 7.55-7.41(m, 3H), 7.40-7.32 (m, 2H), 8.28 (s, 1H), 7.23-7.17 (m, 1H), 6.97 (s,2H), 6.94 (d, 1H), 6.6 (s, 1H), 6.60 (dd, 1H), 5.07 (m, 1H), 5.00-4.91(m, 4H), 4.17-4.02 (m, 2H), 3.96-3.85 (m, 2H), 3.85-3.76 (m, 2H), 3.71(t, 2H), 3.64-3.56 (m, 4H), 3.34-3.12 (m, 10H), 3.01 (m, 2H), 2.33 (t,2H), 2.24-2.12 (m, 2H), 2.09 (s, 3H), 1.70 (p, 2H), 1.45-0.88 (m, 12H),0.88-0.77 (m, 6H); MS (ESI) m/e 1434.2 (M+Na)⁺.

2.87 Synthesis of4-({[(4-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](3-carboxypropyl)amino}piperidin-1-yl)carbonyl]oxy}methyl)-3-[2-(2-{[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]amino}ethoxy)ethoxy]phenylbeta-D-glucopyranosiduronic Acid (Synthon MA) 2.87.13-(1-((3-(2-((1-(((2-(2-(2-aminoethoxy)ethoxy)-4-(((2S,3R,4S,5S,6S)-6-carboxy-3,45-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)piperidin-4-yl)(3-carboxypropyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid

A solution of Example 1.42 (0.050 g) and Example 2.62.6 (0.050 g) inN,N-dimethylformamide (0.5 mL) was treated withN,N-diisopropylethylamine (0.042 mL), and the reaction was stirred atroom temperature for 2 hours. The reaction was concentrated, and theresidue was dissolved in methanol (0.5 mL) and tetrahydrofuran (0.5 mL)and treated with lithium hydroxide hydrate (0.031 g) as a solution inwater (0.5 mL). The reaction was stirred for 1.5 hours and diluted withN,N-dimethylformamide (1 mL). The mixture was purified by reverse phaseHPLC using a Gilson system, eluting with 10-80% acetonitrile in watercontaining 0.1% v/v trifluoroacetic acid. The desired fractions werecombined and freeze-dried to provide the title compound. MS (ESI) m/e1345.7 (M+H)⁺.

2.87.24-({[(4-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](3-carboxypropyl)amino}piperidin-1-yl)carbonyl]oxy}methyl)-3-[2-(2-{[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]amino}ethoxy)ethoxy]phenylbeta-D-glucopyranosiduronic Acid

A solution of Example 2.87.1 (0.047 g) and 2,5-dioxopyrrolidin-1-yl3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate (0.011 g) inN,N-dimethylformamide (0.5 mL) was treated withN,N-diisopropylethylamine (0.031 mL), and the reaction was stirred atroom temperature for 2 hours. The reaction was diluted with a 1:1mixture of N,N-dimethylformamide:water (2 mL). The mixture was purifiedby reverse phase HPLC using a Gilson system, eluting with 10-85%acetonitrile in water containing 0.1% v/v trifluoroacetic acid. Thedesired fractions were combined and freeze-dried to provide the titlecompound. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm 12.87 (s, 1H),8.96 (s, 1H), 8.15-8.07 (m, 2H), 7.88 (d, J=8.1 Hz, 1H), 7.71 (d, J=7.5Hz, 1H), 7.62-7.50 (m, 3H), 7.50-7.45 (m, 1H), 7.45-7.42 (m, 1H), 7.37(s, 1H), 7.33-7.27 (m, 1H), 7.07 (s, 2H), 7.07-7.02 (m, 1H), 6.80-6.74(m, 1H), 6.72-6.66 (m, 1H), 5.23-5.14 (m, 1H), 5.13-5.00 (m, 4H),4.27-4.12 (m, 4H), 4.06-3.95 (m, 4H), 3.92 (s, 2H), 3.83-3.78 (m, 2H),3.57-3.32 (m, 10H), 3.32-3.14 (m, 4H), 3.14-3.06 (m, 2H), 2.90 (s, 2H),2.49-2.37 (m, 4H), 2.19 (s, 3H), 2.12-2.01 (m, 2H), 2.02-1.88 (m, 2H),1.74-1.57 (m, 2H), 1.52 (s, 2H), 1.45-1.30 (m, 4H), 1.30-1.05 (m, 6H),0.95 (s, 6H); MS (ESI) m/e 1495.4 (M+H)⁺.

2.88 Synthesis of4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](3-sulfopropyl)carbamoyl}oxy)methyl]-3-[2-(2-{[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]amino}ethoxy)ethoxy]phenylbeta-D-glucopyranosiduronic Acid (Synthon MD) 2.88.13-(1-((3-(2-((((2-(2-(2-aminoethoxy)ethoxy)-4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)-3-sulfopropyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid

A solution of Example 1.6 (0.039 g) and Example 2.62.6 (0.041 g) inN,N-dimethylformamide (0.5 mL) was treated withN,N-diisopropylethylamine (0.035 mL), and the reaction was stirred atroom temperature for 2 hours. The reaction was concentrated, and theresidue was dissolved in methanol (0.5 mL) and tetrahydrofuran (0.5 mL)and treated with lithium hydroxide hydrate (0.025 g) as a solution inwater (0.5 mL). The reaction was stirred for 1.5 hours and diluted withN,N-dimethylformamide (1 mL). The mixture was purified by reverse phaseHPLC using a Gilson system, eluting with 10-80% acetonitrile in watercontaining 0.1% v/v trifluoroacetic acid. The desired fractions werecombined and freeze-dried to provide the title compound. MS (ESI) m/e1297.8 (M+H)⁺.

2.88.24-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](3-sulfopropyl)carbamoyl}oxy)methyl]-3-[2-(2-{[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]amino}ethoxy)ethoxy]phenylbeta-D-glucopyranosiduronic Acid

To a solution of Example 2.88.1 (0.024 g) and 2,5-dioxopyrrolidin-1-yl3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate (6.40 mg) inN,N-dimethylformamide (0.5 mL) was added N,N-diisopropylethylamine(0.016 mL), and the reaction was stirred at room temperature for 1 hour.The reaction was diluted with a 1:1 mixture ofN,N-dimethylformamide:water (2 mL). The mixture was purified by reversephase HPLC using a Gilson system, eluting with 10-80% acetonitrile inwater containing 0.1% v/v trifluoroacetic acid. The desired fractionswere combined and freeze-dried to provide the title compound. ¹H NMR(500 MHz, dimethyl sulfoxide-d₆) δ ppm 12.87 (s, 1H), 8.09-8.02 (m, 2H),7.79 (d, 1H), 7.61 (d, 1H), 7.52 (dd, 1H), 7.50-7.42 (m, 2H), 7.40-7.33(m, 2H), 7.31 (s, 1H), 7.20 (t, 1H), 6.98 (s, 3H), 6.66 (s, 1H), 6.60(dd, 1H), 5.06 (t, 1H), 4.96 (s, 4H), 4.10 (dq, 4H), 3.81 (d, 4H), 3.71(t, 2H), 3.59 (t, 2H), 3.51-3.35 (m, 4H), 3.26 (td, 6H), 3.17 (q, 2H),3.01 (t, 2H), 2.35 (dt, 4H), 2.10 (d, 3H), 1.75 (d, 2H), 1.44-0.88 (m,12H), 0.82 (d, 6H); MS (ESI) m/e 1446.4 (M−H)⁻.

2.89 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-[4-({[(3-{([2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)amino}azetidin-1-yl)carbonyl]oxy}methyl)phenyl]-N-carbamoyl-L-ornithinamide(Synthon MG)

A solution of Example 1.60 (0.026 g),4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl) carbonate (0.024 g) and N,N-diisopropylethylamine (0.022mL) were stirred together in N,N-dimethylformamide (0.8 mL) at roomtemperature for 3 hours. The reaction was diluted with a 1:1 mixture ofN,N-dimethylformamide:water (2 mL). The mixture was purified by reversephase HPLC using a Gilson system, eluting with 10-80/acetonitrile inwater containing 0.1% v/v trifluoroacetic acid. The desired fractionswere combined and freeze-dried to provide the title compound. ¹H NMR(400 MHz, dimethyl sulfoxide-d₆) δ ppm 12.85 (s, 1H), 9.99 (s, 1H), 8.06(d, 1H), 8.03 (d, 1H), 7.79 (dd, 2H), 7.60 (dd, 3H), 7.55-7.41 (m, 3H),7.36 (td, 2H), 7.29 (t, 3H), 6.99 (s, 2H), 6.95 (d, 1H), 5.99 (s, 1H),5.04-4.92 (m, 4H), 4.37 (q, 1H), 4.34-4.24 (m, 1H), 4.24-4.10 (m, 4H),3.88 (t, 2H), 3.82 (s, 2H), 3.40-3.29 (m, 4H), 3.01 (t, 2H), 2.99-2.91(m, 1H), 2.87 (t, 2H), 2.25-2.06 (m, 5H), 1.95 (dt, 1H), 1.68 (s, 1H),1.60 (s, 1H), 1.54-1.24 (m, 12H), 1.24-0.94 (m, 9H), 0.90-0.78 (m, 12H);MS (ESI) m/e 1507.4 (M+H)⁺.

2.90 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{[26-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-8,24-dioxo-3-(2-sulfoethyl)-11,14,17,20-tetraoxa-3,7,23-triazahexacos-1-yl]oxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (Synthon MS)

To a mixture of Example 1.61.2 (15 mg) and 2,5-dioxopyrrolidin-1-yl1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-oxo-7,10,13,16-tetraoxa-4-azanonadecan-19-oate(16.91 mg) in N,N-dimethylformamide (0.8 mL) was addedN,N-diisopropylethylamine (28.8 μL) at 0° C. The mixture was stirred for3 hours and purified by reverse phase HPLC, using a Gilson system andC18 column, eluting with 20-60% acetonitrile in water containing 0.1%trifluoroacetic acid, to provide the title compound. ¹H NMR (400 MHz,dimethyl sulfoxide-d₆) δ ppm 12.87 (s, 1H), 8.98 (s, 1H), 8.08-7.92 (m,3H), 7.79 (d, 1H), 7.62 (d, 1H), 7.57-7.41 (m, 3H), 7.36 (td, 2H), 7.29(s, 1H), 7.04-6.92 (m, 3H), 4.96 (s, 2H), 3.89 (t, 2H), 3.83 (s, 2H),3.48 (d, 4H), 3.44-3.17 (m, 3H), 3.18-2.83 (m, 10H), 2.38-2.24 (m, 4H),2.11 (s, 3H), 1.78 (m, 2H), 1.50-0.94 (m, 12H), 0.86 (s, 6H). MS (ESI)m/e 1309.3 (M−H).

2.91 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-[4-({[(3-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)amino}propyl)carbamoyl]oxy}methyl)phenyl]-N⁵-carbamoyl-L-ornithinamide(Synthon MR)

To a mixture of Example 1.61.2 (12.8 mg) and4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl) carbonate (10.4 mg) in N,N-dimethylformamide (0.5 mL) at0° C. was added N,N-diisopropylethylamine (24.54 sL). The mixture wasstirred for 3 hours and purified by reverse phase HPLC using a Gilsonsystem and a C18 column, eluting with 20-6/o acetonitrile in watercontaining 0.1% trifluoroacetic acid, to provide the title compound. ¹HNMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm 12.85 (s, 1H), 9.97 (s, 1H),8.97 (s, 1H), 8.04 (t, 2H), 7.79 (dd, 2H), 7.65-7.40 (m, 7H), 7.36 (td,3H), 7.28 (d, 3H), 6.99 (s, 2H), 6.95 (d, 1H), 5.98 (s, 1H), 4.95 (d,4H), 4.49-4.30 (m, 1H), 4.24-4.11 (m, 1H), 3.88 (t, 2H), 3.82 (s, 2H),3.36 (t, 3H), 3.18-2.84 (m, 9H), 2.25-1.88 (m, 5H), 1.85-0.90 (m, 14H),0.91-0.75 (m, 13H). MS (ESI) me (M+H).

2.92 Synthesis ofN-{6-[(iodoacetyl)amino]hexanoyl}-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide(Synthon MQ)

To a mixture of Example 1.2.9 (8.2 mg) and 2,5-dioxopyrrolidin-1-yl6-(2-iodoacetamido)hexanoate (4.7 mg) in N,N-dimethylformamide (0.3 mL)in an ice-bath was added N,N-diisopropylethylamine (3 μL). The mixturewas stirred at 0° C. for 1.5 hours. The reaction was diluted withdimethyl sulfoxide, and the mixture purified by reverse phase HPLC usinga Gilson system and a C18 column, eluting with 20-60% acetonitrile inwater containing 0.1% trifluoroacetic acid, to provide the titlecompound. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm 12.87 (s, 1H),10.00 (s, 1H), 8.21 (d, 1H), 8.06 (dd, 2H), 7.81 (dd, 2H), 7.60 (t, 3H),7.48 (ddd, 3H), 7.36 (td. 2H), 7.28 (d, 3H), 6.95 (d, 1H), 4.97 (d, 4H),4.39 (q, 1H), 4.19 (t, 1H), 3.88 (t, 2H), 3.80 (d, 2H), 3.25 (d, 2H),2.97 (dq. 6H), 2.63 (s, 2H), 2.25-1.88 (m, 5H), 1.78-0.70 (m, 29H). MS(ESI) m/e 1538.4 (M−H).

2.93 Synthesis ofN-{6-[(ethenysulfonyl)amino]hexanoyl}-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide(Synthon MZ) 2.93.1 Methyl 6-(vinylsulfonamido)hexanoate

To a solution of 6-methoxy-6-oxohexan-1-aminium chloride (0.3 g) andtriethylamine (1.15 mL) in dichloromethane at 0° C. was addedethenesulfonyl chloride (0.29 g) dropwise. The reaction mixture waswarmed to room temperature and stirred for 1 hour. The mixture wasdiluted with dichloromethane and washed with brine. The organic layerwas dried over sodium sulfate, filtered, and concentrated to provide thetitle compound. MS (ESI) m/e 471.0 (2M+H)⁺.

2.93.2 6-(vinylsulfonamido)hexanoic Acid

A solution of Example 2.93.1 (80 mg) and lithium hydroxide monohydrate(81 mg) in a mixture of tetrahydrofuran (1 mL) and water (1 mL) wasstirred for 2 hours, then diluted with water (20 mL), and washed withdiethyl ether (10 mL). The aqueous layer was acidified to pH 4 with 1Naqueous HCl and extracted with dichloromethane (3×10 mL). The organiclayer was washed with brine (5 mL), dried over sodium sulfate, filteredand concentrated to provide the title compound.

2.93.3 2,5-dioxopyrrolidin-1-yl 6-(vinylsulfonamido)hexanoate

A mixture of Example 2.93.2 (25 mg),1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide hydrochloride (43.3 mg)and 1-hydroxypyrrolidine-2,5-dione (15.6 mg) in dichloromethane (8 mL)was stirred overnight, washed with saturated aqueous ammonium chloridesolution and brine, and concentrated to provide the title compound.

2.93.4N-{6-[(ethenylsulfonyl)amino]hexanoyl}-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1,13,7]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N5-carbamoyl-L-ornithinamide

The title compound was prepared as described in Example 2.83, replacingExample 1.2.9 and 2,5-dioxopyrrolidin-1-yl6-(2-chloroacetamido)hexanoate with Example 2.66.1 and Example 2.93.3,respectively. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm 12.85 (s,1H), 9.98 (s, 1H), 8.05 (dd, 2H), 7.79 (d, 2H), 7.60 (t, 3H), 7.55-7.40(m, 3H), 7.36 (td, 2H), 7.27 (d, 3H), 7.19 (t, 1H), 6.95 (d, 1H), 6.66(dd, 1H), 6.09-5.90 (m, 2H), 4.97 (d, 4H), 4.39 (q, 1H), 4.20 (t, 1H),3.88 (t, 2H), 3.80 (d, 2H), 3.25 (d, 2H), 2.97 (dt, 4H), 2.78 (q, 2H),2.64 (q, 2H), 2.22-1.86 (m, 6H), 1.77-0.89 (m, 16H), 0.89-0.72 (m, 12H).MS (ESI) m/e 1460.6 (M−H).

2.94 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-(1-{[3-(2-{[3-({6-[(iodoacetyl)amino]hexanoyl}amino)propyl](2-sulfoethyl)amino}ethoxy)-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl]methyl}-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylicAcid (Synthon NA)

The title compound was prepared using the procedure in Example 2.83,replacing Example 1.2.9 and 2,5-dioxopyrrolidin-1-yl6-(2-chloroacetamido)hexanoate with Example 2.61.2 and2,5-dioxopyrrolidin-1-yl 6-(2-iodoacetamido)hexanoate, respectively. ¹HNMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm 12.87 (s, 1H), 8.98 (s, 1H),8.20 (t, 1H), 8.04 (d, 1H), 7.91 (t, 1H), 7.79 (d, 1H), 7.62 (d, 1H),7.53 (d, 1H), 7.50-7.41 (m, 2H), 7.36 (td, 2H), 7.29 (s, 1H), 6.96 (d,1H), 4.96 (s, 2H), 3.89 (t, 2H), 3.83 (s, 2H), 3.06 (dt, 8H), 2.89 (t,2H), 2.17-1.99 (m, 5H), 1.76 (s, 2H), 1.56-0.93 (m, 14H), 0.86 (s, 6H).MS (ESI) m/e 1190.3 (M−H).

2.95 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydriosoquinolin-2(1H)-yl]-3-{1-[(3-{2-[(3-{[6-(ethenylsulfonyl)hexanoyl]amino}propyl)(2-sulfoethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (Synthon NB)

The title compound was prepared using the procedure in Example 2.83,replacing Example 1.2.9 and 2,5-dioxopyrrolidin-1-yl6-(2-chloroacetamido)hexanoate with Example 1.61.2 and Example 2.82.5,respectively. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm 12.87 (s,1H), 8.98 (s, 1H), 8.04 (d, 1H), 7.92 (t, 1H), 7.79 (d, 1H), 7.62 (d,1H), 7.53 (d, 1H), 7.51-7.41 (m, 2H), 7.36 (td, 2H), 7.29 (s, 1H),7.01-6.90 (m, 2H), 6.29-6.16 (m, 2H), 4.96 (s, 2H), 3.89 (t, 2H), 3.83(s, 2H), 3.45-3.19 (m, 2H), 3.19-2.95 (m, 8H), 2.89 (t, 2H), 2.16-1.98(m, 5H), 1.84-1.66 (m, 2H), 1.64-1.21 (m, 13H), 1.08 (dq, 6H), 0.86 (s,6H). MS (ESI) m/e 1199.3 (M+H).

2.96 Synthesis ofN-[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methy]phenyl}-N⁵-carbamoyl-L-ornithinamide(Synthon NP) 2.96.1 (S)-(9H-fluoren-9-yl)methyl(1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)arbamate

(S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanoic acid(40 g) was dissolved in dichloromethane (1.3L). (4-Aminophenyl)methanol(13.01 g).2-(3H-[1.2.3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisouroniumhexafluorophosphate(V) (42.1 g) and N,N-diisopropylethylamine (0.035 L)were added to the solution, and the resulting mixture was stirred atroom temperature for 16 hours. The product was collected by filtrationand rinsed with dichloromethane. The combined solids were dried undervacuum to yield the title compound, which was used in the next stepwithout further purification. MS (ESI) m/e 503.3 (M+H)⁺.

2.96.2 (S)-2-amino-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide

Example 2.96.1 (44 g) was dissolved in N,N-dimethylformamide (300 mL).The solution was treated with diethylamine (37.2 mL) and stirred for onehour at room temperature. The reaction mixture was filtered, and thesolvent was concentrated under reduced pressure. The crude product waspurified by basic alumina chromatography eluting with a gradient of0-30% methanol in ethyl acetate to give the title compound. MS (ESI) m/e281.2 (M+H)⁺.

2.96.3 Tert-butyl((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)arbamate

(S)-2-(Tert-butoxycarbonylamino)-3-methylbutanoic acid (9.69 g) wasdissolved in N,N-dimethylformamide (200 mL). To the solution was added2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisouroniumhexafluorophosphate(V) (18.65 g), and the reaction was stirred for onehour at room temperature. Example 2.96.2 (12.5 g) andN,N-diisopropylethylamine (15.58 mL) were added and the reaction mixturewas stirred for 16 hours at room temperature. The solvent wasconcentrated under reduced pressure and the residue was purified bysilica gel chromatography, eluting with 10% methanol in dichloromethane,to give the title compound. MS (ESI) m/e 480.2 (M+H)⁺.

2.96.4(S)-2-((S)-2-amino-3-methylbutanamido)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide

Example 2.96.3 (31.8 g) was dissolved in dichloromethane (650 mL) andtrifluoroacetic acid (4.85 mL) was added to the solution. The reactionmixture was stirred for three hours at room temperature. The solvent wasconcentrated under reduced pressure to yield a mixture of the crudetitle compound and4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl2,2,2-trifluoroacetate. The crude material was dissolved in a 1:1dioxane/water solution (300 mL) and to the solution was added sodiumhydroxide (5.55 g). The mixture was stirred for three hours at roomtemperature. The solvent was concentrated under vacuum, and the crudeproduct was purified by reverse phase HPLC using a CombiFlash system,eluting with a gradient of 5-60/acetonitrile in water containing 0.05%v/v ammonium hydroxide, to give the title compound. MS (ESI) m/e 380.2(M+H)⁺.

2.96.5(S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3-methylbutanamido)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide

To a solution of Example 2.96.4 (38 mg) in N,N-dimethylformamide (1 mL)was added 2,5-dioxopyrrolidin-1-yl3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate (26.7 mg). Thereaction mixture was stirred at room temperature overnight and purifiedby reverse phase HPLC using a Gilson system, eluting with a gradient of10-85% acetonitrile in water containing 0.1% v/v trifluoroacetic acid,to give the title compound. MS (ESI) m/e 531.06 (M+H)⁺.

2.96.64-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl) Carbonate

To a solution of Example 2.96.5 (53.1 mg) in N,N-dimethylformamide (3mL) was added bis(4-nitrophenyl) carbonate (60.8 mg). The reactionmixture was stirred at room temperature overnight and purified byreverse phase HPLC using a Gilson system, eluting with a gradient of10-85% acetonitrile in water containing 0.1% v/v trifluoroacetic acid,to give the title compound. MS (ESI) m/e 696.2 (M+H)⁺.

2.96.7N-[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1,13,7]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N5-carbamoyl-L-ornithinamide

The title compound was prepared as described in Example 2.1, replacingExample 1.2.9 and4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl) carbonate with Example 1.24.2 and Example 2.96.6,respectively. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm 9.91 (s,1H), 9.80 (s, 2H), 8.33 (s, 2H), 7.% (s, 2H), 7.81 (d, 4H), 7.61 (s,2H), 7.43 (d, 10H), 7.34-7.02 (m, 14H), 5.92 (s, 8H), 4.94-4.70 (m, 6H),4.18 (d, 11H), 3.85 (s, 8H), 3.05-2.66 (m, 8H), 2.30-2.13 (m, 14H),2.03-1.49 (m, 2H), 0.92-0.63 (m, 40H). MS (ESI) m/e 1408.3 (M−H)⁺.

2.97 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydriosoquinolin-2(1H)-yl]-3-{1-[(3-{2-[(2-carboxyethyl){([2-{[(2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl]oxy}-4-[2-(2-{[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]amino}ethoxy)ethoxy]benzyl)oxy]carbonyl}amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (Synthon NN) 2.97.14-(2-(2-bromoethoxy)ethoxy)-2-hydroxybenzaldehyde

A solution of 2,4-dihydroxybenzaldehyde (1.0 g).1-bromo-2-(2-bromoethoxy)ethane (3.4 g) and potassium carbonate (1.0 g)in acetonitrile (30 mL) was heated to 75° C. for 2 days. The reactionwas cooled, diluted with ethyl acetate (100 mL), washed with water (50mL) and brine (50 mL), dried over magnesium sulfate, filtered andconcentrated. Purification of the residue by silica gel chromatography,eluting with a gradient of 5-30% ethyl acetate in heptane, provided thetitle compound. MS (ELSD) m/e 290.4 (M+H)⁺.

2.97.2 4-(2-(2-azidoethoxy)ethoxy)-2-hydroxybenzaldehyde

To a solution of Example 2.97.1 (1.26 g) in N,N-dimethylformamide (10mL) was added sodium azide (0.43 g), and the reaction was stirred atroom temperature overnight. The reaction was diluted with diethyl ether(100 mL), washed with water (50 mL) and brine (50 mL), dried overmagnesium sulfate, filtered, and concentrated. Purification of theresidue by silica gel chromatography, eluting with a gradient of 5-30%ethyl acetate in heptane, gave the title compound. MS (ELSD) m/e 251.4(M+H)⁺.

2.97.3(2S,3R,4S,5S,6S)-2-(5-(2-(2-azidoethoxy)ethoxy)-2-formylphenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate

A solution of Example 2.97.2 (0.84 g).(3R,4S,5S,6S)-2-bromo-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (1.99 g) and silver (I) oxide (1.16 g) were stirred togetherin acetonitrile (15 mL). After stirring overnight, the reaction wasdiluted with dichloromethane (20 mL). Diatomaceous earth was added, andthe reaction filtered and concentrated. Purification of the residue bysilica gel chromatography, eluting with a gradient of 5-75% ethylacetate in heptane, gave the title compound.

2.97.4(2S,3R,4S,5S,6S)-2-(5-(2-(2-azidoethoxy)ethoxy)-2-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate

A solution of Example 2.97.3 (0.695 g) in methanol (5 mL) andtetrahydrofuran (2 mL) was cooled to 0° C. Sodium borohydride (0.023 g)was added, and the reaction was warmed to room temperature. Afterstirring for a total of 1 hour, the reaction was poured into a mixtureof ethyl acetate (75 mL) and water (25 mL), and saturated aqueous sodiumbicarbonate (10 mL) was added. The organic layer was separated, washedwith brine (50 mL), dried over magnesium sulfate, filtered, andconcentrated. Purification of the residue by silica gel chromatography,eluting with a gradient of 5-85% ethyl acetate in heptane, gave thetitle compound. MS (ELSD) m/e 551.8 (M−H₂O)⁻.

2.97.5(2S,3R,4S,5S,6S)-2-(5-(2-(2-aminoethoxy)ethoxy)-2-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate

To Example 2.97.4 (0.465 g) in tetrahydrofuran (20 mL) was added 5% Pd/C(0.1 g) in a 50 mL pressure bottle, and the mixture was shaken for 16hours under 30 psi hydrogen. The reaction was filtered and concentratedto give the title compound, which was used without further purification.MS (ELSD) me 544.1 (M+H)⁺.

2.97.6(2S,3R,4S,5S,6S)-2-(5-(2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethoxy)ethoxy)-2-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate

A solution of Example 2.97.5 (0.443 g) in dichloromethane (8 mL) wascooled to 0° C. then N,N-diisopropylethylamine (0.214 mL) and(9H-fluoren-9-yl)methyl carbonochloridate (0.190 g) were added. After 1hour, the reaction was concentrated. Purification of the residue bysilica gel chromatography, eluting with a gradient of 5-95% ethylacetate in heptane, gave the tide compound. MS (ELSD)/e 748.15 (M-OH)⁻.

2.97.7(2S,3R,4S,5S,6S)-2-(5-(2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethoxy)ethoxy)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate

To a solution of Example 2.97.6 (0.444 g) in N,N-dimethylformamide (5mL) was added N,N-diisopropylethylamine (0.152 mL) andbis(4-nitrophenyl) carbonate (0.353 g), and the reaction was stirred atroom temperature. After 5 hours, the reaction was concentrated.Purification of the residue by silica gel chromatography, eluting with agradient of 5-90% ethyl acetate in heptane, gave the title compound.

2.97.83-(1-((3-(2-((((4-(2-(2-aminoethoxy)ethoxy)-2-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)(2-carboxyethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid, Trifluoroacetic Acid Salt

To a solution of Example 1.25 (0.070 g) and Example 2.97.7 (0.070 g) inN,N-dimethylformamide (0.4 mL) was added N,N-diisopropylethylamine(0.066 mL). After stirring overnight, the reaction was concentrated. Theresidue was dissolved in tetrahydrofuran (0.75 mL) and methanol (0.75mL), and lithium hydroxide monohydrate (0.047 g) was added as a solutionin water (0.75 mL). After 3 hours, the reaction was diluted withN,N-dimethylformamide (1 mL) and quenched with trifluoroacetic acid(0.116 mL). The mixture was purified by reverse phase HPLC using aGilson system, eluting with 10-75% acetonitrile in water containing 0.1%v/v trifluoroacetic acid. The desired fractions were combined andfreeze-dried to provide the title compound.

2.97.96-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-(2-((((2-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-4-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)ethoxy)ethoxy)benzyl)oxy)carbonyl)(2-carboxyethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinicAcid

A solution of Example 2.97.8 (0.027 g), 2,5-dioxopyrrolidin-1-yl3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate (7.92 mg) andN,N-diisopropylethylamine (0.017 mL) were stirred together inN,N-dimethylformamide (0.4 mL) for 1 hour. The reaction was quenchedwith a 1:1 mixture of water and N,N-dimethylformamide (2 mL). Themixture was purified by reverse phase HPLC using a Gilson system,eluting with 10-75% acetonitrile in water containing 0.1% v/vtrifluoroacetic acid. The desired fractions were combined andfreeze-dried to provide the title compound. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 12.81 (s, 1H), 8.03 (d, 2H), 7.79 (d, 1H), 7.62 (d,1H), 7.54-7.40 (m, 3H), 7.36 (td, 2H), 7.28 (s, 1H), 7.18 (d, 1H), 6.98(s, 2H), 6.95 (d, 1H), 6.69 (d, 1H), 6.60 (d, 1H), 5.03 (d, 3H), 4.96(s, 2H), 4.05 (s, 2H), 3.93 (d, 2H), 3.88 (t, 2H), 3.80 (d, 2H),3.75-3.67 (m, 2H), 3.59 (t, 6H), 3.29 (q, 6H), 3.17 (q, 2H), 3.01 (t,2H), 2.47 (d, 2H), 2.33 (t, 2H), 2.09 (s, 3H), 1.44-0.88 (m, 12H), 0.82(d, 6H): MS (ESI) m/e 1396.5 (M−H)⁻.

2.98 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-3-sulfo-L-alanyl-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1J]dec-1-yl}oxy)ethyl](2-carboxyethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide(Synthon NO) 2.98.13-(1-((3-(2-((((4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)(2-carboxyethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid

A solution of Example 1.25.2 (0.059 g), (9H-fluoren-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-1-oxobutan-2-yl)carbamate(0.053 g) and N,N-diisopropylethylamine (0.055 mL) inN,N-dimethylformamide (0.5 mL) was stirred at room temperatureovernight. Diethylamine (0.066 mL) was added to the reaction, andstirring was continued for 30 minutes. The reaction was diluted with a1:1 mixture of N,N-dimethylformamide and water (2 mL) and quenched bythe addition of trifluoroacetic acid (0.073 mL). The mixture waspurified by reverse phase HPLC using a Gilson system, eluting with10-75% acetonitrile in water containing 0.1% v/v trifluoroacetic acid.The desired fractions were combined and freeze-dried to provide thetitle compound. MS (ESI) m/e 1223.8 (M+H)⁺.

2.98.23-(1-((3-(2-((((4-((S)-2-((S)-2-((R)-2-amino-3-sulfopropanamido)-3-methylbutanamido)-5-ureidopentanamido)bezyl)oxy)carbonyl)(2-carboxyethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid, Trifluoracetic Acid Salt

A solution of(R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-sulfopropanoic acid(0.021 g), O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (0.020 g) and N,N-diisopropylethylamine (0.031 mL)in N,N-dimethylformamide (0.4 mL) was stirred for 3 minutes. Thesolution was added to Example 2.98.1 (0.043 g) as a solution inN,N-dimethylformamide (0.4 mL). After stirring for 30 minutes, asolution of lithium hydroxide monohydrate (0.022 g) in water (0.5 mL)was added, and the reaction was stirred for 1 hour. The reaction wasdiluted with a 1:1 mixture of N,N-dimethylformamide and water (2 mL) andquenched by the addition of trifluoroacetic acid (0.054 mL). The mixturewas purified by reverse phase HPLC using a Gilson system, eluting with10-75% acetonitrile in water containing 0.1% v/v trifluoroacetic acid.The desired fractions were combined and freeze-dried to provide thetitle compound. MS (ESI) m/e 1376.5 (M+1).

2.98.36-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-(2-((2-carboxyethyl)(((4-((S)-2-((S)-2-((R)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-sulfopropanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinicAcid

A solution of Example 2.98.2 (0.025 g), 2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (7.77 mg) andN,N-diisopropylethylamine (0.015 mL) in N,N-dimethylformamide (0.4 mL)was stirred for 1 hour. The reaction was diluted with a 1:1 mixture ofwater and N,N-dimethylformamide (2 mL). The mixture was purified byreverse phase HPLC using a Gilson system, eluting with 10-75%acetonitrile in water containing 0.1% v/v trifluoroacetic acid. Thedesired fractions were combined and freeze-dried to provide the titlecompound. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm 12.85 (s, 1H),9.46 (s, 1H), 8.20 (d, 1H), 8.07 (d, 1H), 8.03 (d, 1H), 8.00 (d, 1H),7.79 (d, 1H), 7.69 (d, 2H), 7.61 (d, 1H), 7.51 (d, 1H), 7.49-7.45 (m,1H), 7.43 (d, 1H), 7.36 (td, 2H), 7.29 (s, 1H), 7.25 (d, 2H), 6.97 (s,2H), 6.95 (d, 1H), 4.98 (s, 2), 4.96 (s, 2H), 4.73 (s, 2H), 4.16 (s,2H), 4.03 (dd, 2H), 3.88 (t, 2H), 3.81 (s, 2H), 3.51-3.32 (m, 6H), 3.28(t, 2H), 3.09 (dd, 1H), 3.06-2.94 (m, 4H), 2.89 (dd, 1H), 2.46 (d, 2H),2.16 (dd, 1H), 2.09 (d, 4H), 1.74 (s, 2H), 1.62-1.29 (m, 8H), 1.29-0.92(m, 12H), 0.92-0.78 (m, 12H). MS (ESI) m/e 1566.6 (M−H)⁻.

2.99 Synthesis of Control Synthon4-[([2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl)-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](methyl)carbamoyl}oxy)methyl]-2-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-beta-alanyl}amino)phenylbeta-D-glucopyranosidumnic Acid (Synthon H) 2.99.1(2S,3R,4S,5S,6S)-2-(4-formyl-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate

To a solution of(2R,3R,4S,5S,6S)-2-bromo-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (4 g) in acetonitrile (100 mL)) was added silver(I) oxide(10.04 g) and 4-hydroxy-3-nitrobenzaldehyde (1.683 g). The reactionmixture was stirred for 4 hours at room temperature and filtered. Thefiltrate was concentrated, and the residue was purified by silica gelchromatography, eluting with 5-50% ethyl acetate in heptanes, to providethe title compound. MS (ESI) m/e (M+18)⁺.

2.99.2(2S,3R,4S,5S,6S)-2-(4-(hydroxymethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate

To a solution of Example 2.99.1 (6 g) in a mixture of chloroform (75 mL)and isopropanol (18.75 mL) was added 0.87 g of silica gel. The resultingmixture was cooled to 0° C. NaBH₄ (0.470 g) was added, and the resultingsuspension was stirred at 0° C. for 45 minutes. The reaction mixture wasdiluted with dichloromethane (100 mL) and filtered through diatomaceousearth. The filtrate was washed with water and brine and concentrated togive the crude product, which was used without further purification. MS(ESI) m/e (M+NH₄)⁺:

2.99.3(2S,3R,4S,5S,6S)-2-(2-amino-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate

A stirred solution of Example 2.99.2 (7 g) in ethyl acetate (81 mL) washydrogenated at 20° C. under 1 atmosphere H₂, using 10% Pd/C (1.535 g)as a catalyst for 12 hours. The reaction mixture was filtered throughdiatomaceous earth, and the solvent was evaporated under reducedpressure. The residue was purified by silica gel chromatography, elutingwith 95/5 dichloromethane/methanol, to give the title compound.

2.99.4 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoic Acid

3-Aminopropanoic acid (4.99 g) was dissolved in 10% aqueous Na₂CO₃solution (120 mL) in a 500 mL flask and cooled with an ice bath. To theresulting solution, (9H-fluoren-9-yl)methyl carbonochloridate (14.5 g)in 1,4-dioxane (100 mL) was gradually added. The reaction mixture wasstirred at room temperature for 4 hours, and water (800 mL) was thenadded. The aqueous phase layer was separated from the reaction mixtureand washed with diethyl ether (3×750 mL). The aqueous layer wasacidified with 2N HCl aqueous solution to a pH value of 2 and extractedwith ethyl acetate (3×750 mL). The organic layers were combined andconcentrated to obtain crude product. The crude product wasrecrystallized in a mixed solvent of ethyl acetate: hexane 1:2 (300 mL)to give the title compound.

2.99.5 (9H-fluoren-9-yl)methyl (3-chloro-3-oxopropyl)carbamate

To a solution of Example 2.99.4 in dichloromethane (160 mL) was addedsulfurous dichloride (50 mL). The mixture was stirred at 60° C. for 1hour. The mixture was cooled and concentrated to give the titlecompound.

2.99.6(2S,3R,4S,5S,6S)-2-(2-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate

To a solution of Example 2.99.3 (6 g) in dichloromethane (480 mL) wasadded N,N-diisopropylethylamine (4.60 mL). Example 2.99.5 (5.34 g) wasadded, and the mixture was stirred at room temperature for 30 minutes.The mixture was poured into saturated aqueous sodium bicarbonate and wasextracted with ethyl acetate. The combined extracts were washed withwater and brine and were dried over sodium sulfate. Filtration andconcentration gave a residue that was purified via radialchromatography, using 0-100% ethyl acetate in petroleum ether as mobilephase, to give the title compound.

2.99.7(2S,3R,4S,5S,6S)-2-(2-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate

To a mixture of Example 2.99.6 (5.1 g) in N,N-dimethylformamide (200 mL)was added bis(4-nitrophenyl) carbonate (4.14 g) andN,N-diisopropylethylamine (1.784 mL). The mixture was stirred for 16hours at room temperature and concentrated under reduced pressure. Thecrude material was dissolved in dichloromethane and aspirated directlyonto a 1 mm radial Chromatotron plate and eluted with 50-100% ethylacetate in hexanes to give the title compound. MS (ESI) m/e (M+H)⁺.

2.99.83-(1-((3-(2-((((3-(3-aminopropanamido)-4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)(methyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid

To a solution of Example 1.13.7 (325 mg) and Example 2.99.7 (382 mg) inN,N-dimethylformamide (9 mL) at 0° C. was added N,N-diisopropylamine(49.1 mg). The reaction mixture was stirred at 0° C. for 5 hours, andacetic acid (22.8 mg) was added. The resulting mixture was diluted withethyl acetate and washed with water and brine. The organic layer wasdried over Na₂SO₄, filtered and concentrated. The residue was dissolvedin a mixture of tetrahydrofuran (10 mL) and methanol (5 mL). To thissolution at 0° C. was added 1 M aqueous lithium hydroxide solution (3.8mL). The resulting mixture was stirred at 0° C. for 1 hour, acidifiedwith acetic acid and concentrated. The concentrate was lyophilized toprovide a powder. The powder was dissolved in N,N-dimethylformamide (10mL), cooled in an ice-bath, and piperidine (1 mL) at 0° C. was added.The mixture was stirred at 0° C. for 15 minutes and 1.5 mL of aceticacid was added. The solution was purified by reverse-phase HPLC using aGilson system, eluting with 30-80% acetonitrile in water containing 0.1%v/v trifluoroacetic acid, to provide the title compound. MS (ESI) n/e1172.2 (M+H)⁺.

2.99.94-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1,13,7]dec-1-yl}oxy)ethyl](methyl)carbamoyl}oxy)methyl]-2-{(N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-beta-alanyl}amino)phenylbeta-D-glucopyranosiduronic Acid

To Example 2.99.8 (200 mg) in N,N-dimethylformamide (5 mL) at 0° C. wasadded 2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (105 mg) andN,N-diisopropylethylamine (0.12 mL). The mixture was stirred at 0° C.for 15 minutes, warmed to room temperature and purified by reverse-phaseHPLC on a Gilson system using a 100 g C18 column, eluting with 30-80%acetonitrile in water containing 0.1% v/v trifluoroacetic acid, toprovide the title compound. ¹H NMR (500 MHz, dimethyl sulfoxide-d₆) δppm 12.85 (s, 2H) 9.07 (s, 1H) 8.18 (s, 1H) 8.03 (d, 1f) 7.87 (t, 1H)7.79 (d, 1H) 7.61 (d, 1H) 7.41-7.53 (m, 3H) 7.36 (q, 2H) 7.28 (s, 1H)7.03-7.09 (m, 1H) 6.96-7.03 (m, 3H) 6.94 (d, 1H) 4.95 (s, 4H) 4.82 (t,1H) 3.88 (t, 3H) 3.80 (d, 2H) 3.01 (t. 2H) 2.86 (d, 3H) 2.54 (t, 2H)2.08 (s, 3H) 2.03 (t, 2H) 1.40-1.53 (m, 4H) 1.34 (d, 2H) 0.90-1.28 (m,12H) 0.82 (d, 6H). MS (ESI) m/e 1365.3 (M+H)⁺.

2.100 Synthesis of Control Synthon4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](methyl)carbamoyl}oxy)methyl]-2-({N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecan-1-oyl]-beta-alanyl}amino)phenylbeta-D-glucopyranosiduronic Acid (Synthon I)

The title compound was prepared using the procedure in Example 2.99.9,replacing 2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate with2,5-dioxopyrrolidin-1-yl1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-oxo-7,10,13,16-tetraoxa-4-azanonadecan-19-oate.¹H NMR (500 MHz, dimethyl sulfoxide-d₆) δ ppm 8.95 (s, 1H) 8.16 (s, 1H)7.99 (d, 1H) 7.57-7.81 (m. 4H) 7.38-7.50 (m, 3H) 7.34 (q, 2H) 7.27 (s,1H) 7.10 (d, 1H) 7.00 (d, 1H) 6.88-6.95 (m, 2H) 4.97 (d. 4H) 4.76 (d,2H) 3.89 (t, 2H) 3.84 (d, 2H) 3.80 (s, 2H) 3.57-3.63 (m, 4H) 3.44-3.50(m, 4H) 3.32-3.43 (m, 6H) 3.29 (t, 2H) 3.16 (q, 2H) 3.02 (t, 2H) 2.87(s, 3H) 2.52-2.60 (m, 2H) 2.29-2.39 (m, 3H) 2.09 (s, 3H) 1.37 (s, 2H)1.20-1.29 (m, 4H) 1.06-1.18 (m, 4H) 0.92-1.05 (m, 2H) 0.83 (s, 6H). MS(ESI) m/e 1568.6 (M−H)⁻.

2.101 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{[(43S,46S)-43-({[(4-{[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl)oxy]carbonyl}amino)-46-methyl-37,44,47-trioxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-38,45,48-triazapentacontan-50-yl]oxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (Synthon OK)

The title compound was prepared as described in Example 2.7, replacingExample 1.13.8 with Example 1.66.7. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 12.85 (s, 1H), 8.21-7.97 (m, 4H), 7.79 (d, 4H),7.71-7.32 (m, 15H), 7.28 (t, 4H), 7.02-6.91 (m, 3H), 4.95 (d, 5H),4.33-4.12 (m, 3H), 3.98-3.76 (m, 11H), 3.41-3.21 (m, 22H), 3.21-2.90 (m,12H), 2.24-2.05 (m, 7H), 1.81-0.90 (m, 46H), 0.90-0.78 (m, 17H). MS(ESI) m/e 2014.0 (M+H)⁺, 1007.5 (M+2H), 672.0 (M+3H)³⁺.

2.102 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-(6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-1,2,3,4-tetrahydroquinolin-7-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-carboxyethyl)carbamoyl}oxy)methyl]phenyl)-N⁵-carbamoyl-L-ornithinamide(Synthon OW)

The title compound was prepared as described in Example 2.1, replacingExample 1.2.9 with Example 1.62.5 ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 9.95 (s, 1H), 8.36 (s, 1H), 8.02 (d, 1H), 7.96 (d,1H), 7.88-7.68 (m, 4H), 7.57 (d, 2H), 7.42 (s, 2H), 7.34 (t, 1H), 7.25(dd, 3H), 7.19 (t, 1H), 6.95 (s, 2H), 5.96 (s, 1H), 4.% (s, 2H), 4.35(q, 1H), 4.15 (dd, 1H), 3.93 (t, 2H), 3.83 (d, 2H), 3.32 (t, 2H), 3.27(d, 1H), 2.93 (dtd, 1H), 2.80 (t, 2H), 2.47 (p, 19H), 2.24-2.02 (m 5H),1.91 (p, 3H), 1.74-1.25 (m 8H), 1.27-0.89 (m, 10H), 0.79 (dd, 13H). MS(ESI) m/e 1414.4 (M+H)⁺.

2.103 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-carboxyethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide(Synthon PC)

The title compound was prepared as described in Example 2.1, replacingExample 1.2.9 with Example 1.68.7. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 13.07 (s, 1H), 9.95 (s, 1H), 8.99 (s, 1H), 8.33 (dd,1H), 8.25-8.09 (m, 3H), 8.12-7.95 (m, 3H), 7.90 (d, 1H), 7.76 (dd, 2H),7.73-7.63 (m 1H), 7.56 (s, 3H), 7.41-7.29 (m, 1H), 6.95 (s, 2H), 5.97(s, 1H), 4.96 (s, 2H), 4.35 (d. 2H), 4.15 (dd, 1H), 3.50-3.22 (m, 10H),2.92 (dtd, 3H), 2.29-2.00 (m, 6H), 1.92 (q, 1H), 1.75-0.88 (m, 24H),0.79 (dd, 15H). MS (ESI) m/e 1409.5 (M+H)⁺.

2.104 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-3-{1-[(3-{2-[(2-carboxyethyl){[(2-{[(2R,3S,4R,5R,6R)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl]oxy}-4-[2-(2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}ethoxy)ethoxy]benzyl)oxy]carbonyl}amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (Synthon PI) 2.104.13-(1-((3-(2-((((4-(2-(2-aminoethoxy)ethoxy)-2-(((2R,3S,4R,5R,6R)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)-2-carboxyethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)naphthalen-2-yl)picolinicAcid

To a cold (0° C.) mixture of Example 2.97.7 (26.9 mg) and Example 1.68.7(23.5 mg) in N,N-dimethylformamide (2 mL) was addedN-ethyl-N-isopropylpropan-2-amine (0.043 mL). The reaction was slowlywarmed to room temperature and stirred overnight. LC/MS showed theexpected product as the major peak. To the reaction mixture was addedwater (1 mL) and LiOH H_(Z) (20 mg). The mixture was stirred at roomtemperature for 3 hours. The mixture was diluted withN,N-dimethylformamide (2 mL), filtered and purified by reverse-phaseHPLC on a Gilson system (C18 column), eluting with 20-80% acetonitrilein water containing 0.1% trifluoroacetic acid, to give the titlecompound. MS (ESI) m/e 1242.2 (M−H)⁻.

2.104.26-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-3-{1-[(3-{2-[(2-carboxyethyl){[(2-{[(2R,3S,4R,5R,6R)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl]oxy}-4-[2-(2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}ethoxy)ethoxy]benzyl)oxy]carbonyl}amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.13,7]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid

The title compound was prepared as described in Example 2.97.9 byreplacing Example 2.97.8 with Example 2.104.1 and replacing2,5-dioxopyrrolidin-1-yl3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate with2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate. ¹H NMR (400 MHz,dimethyl sulfoxide-d₆) δ ppm 13.06 (s, 2H), 8.99 (s, 1H), 8.34 (dd. 1H),8.25-8.10 (m, 3H), 8.04 (d, 1H), 7.98 (d, 1H), 7.90 (d, 1H), 7.78 (d,2H), 7.72-7.63 (m, 1H), 7.50-7.42 (m, 2H), 7.34 (t, 1H), 7.16 (d, 1H),6.94 (s, 2H), 6.65 (d, 1H), 6.56 (dd, 1H), 4.02 (t, 2H), 3.90 (d, 1H),3.83 (s, 2H), 3.66 (t, 3H), 3.28 (q, 4H), 3.15 (q, 2H), 2.19 (s, 3H),1.99 (t, 2H), 1.51-1.30 (m, 6H), 1.28-0.88 (m, 11H), 0.81 (d, 6H). MS(ESI) m/e 1433.4 (M+H)⁺.

2.105 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[5-(1,3-benzothiazol-2-ylcarbamoyl)quinolin-3-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N-carbamoyl-L-ornithinamide(Synthon PJ)

The title compound was prepared as described in Example 2.1, replacingExample 1.2.9 with Example 1.69.6. ¹H NMR (500 MHz, dimethylsulfoxide-d₆) δ ppm 13.23 (s, 1H), 9.99 (s, 1H), 9.73 (d, 1H), 9.45 (s,1H), 8.33 (t, 2H), 8.18 (d, 1H), 8.07 (dd, 2H), 8.02 (dd, 1H), 7.97 (dd,1H), 7.80 (t, 2H), 7.65-7.55 (m, 2H), 7.53-7.44 (m, 2H), 7.37 (t, 1H),7.27 (d, 2H), 6.98 (s, 2H), 4.98 (d, 2H), 4.38 (d, 1H), 4.18 (d, 1H),3.56-3.31 (m, 3H), 3.26 (d, 2H), 3.08-2.89 (m, 2H), 2.64 (t, 2H), 2.23(d, 3H), 2.12 (dp, 2H), 1.95 (s, 1f). 1.68 (s, 1H), 1.62-1.29 (m, 7H),1.29-0.90 (m 9H), 0.90-0.74 (m 12H). MS (ESI) m/e 1446.3 (M−H)⁻.

2.106 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[4-(1,3-benzothiazol-2-ylcarbamoyl)quinolin-6-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-carboxyethyl)carbamoyl}oxy)methyl]phenyl}-N³-carbamoyl-L-ornithinamide(Synthon PU)

The title compound was prepared as described in Example 2.1, replacingExample 1.2.9 with Example 1.70. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆)δ ppm 9.97 (s, 1H), 9.12 (d, 1H), 8.93 (s, 1H), 8.60 (dd, 1H), 8.24 (dd,2H), 8.05 (dd, 2H), 7.99-7.87 (m, 2H), 7.78 (dd, 2H), 7.67-7.51 (m, 3H),7.43-7.31 (m, 1H), 7.26 (d, 2H), 6.97 (s, 2H), 5.98 (s, 1H), 4.97 (s,2H), 4.37 (d, 2H), 4.17 (dd, 1H), 3.49-3.22 (m 11H), 2.95 (ddd, 3H),2.20 (s, 4H), 2.19-1.86 (m, 3H), 1.74-0.89 (m, 22H), 0.81 (dd, 15H). MS(ESI) n m/e 1410.4 (M−H)⁻.

2.107 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[4-(1,3-benzothiazol-2-ylcarbamoyl)quinolin-6-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethylticyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide(Synthon PV)

The title compound was prepared as described in Example 2.1, replacingExample 1.2.9 with Example 1.70.5. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 9.96 (s, 1H), 9.11 (d, 1H), 8.92 (s, 1H), 8.60 (dd,1H), 8.23 (dd, 2H), 8.12-7.97 (m, 2H), 7.98-7.92 (m, 2H), 7.77 (dd, 2H),7.56 (t, 2H), 7.51-7.42 (m, 2H), 7.42-7.31 (m, 1H), 7.24 (d, 2H), 6.95(s, 2H), 4.95 (d, 2H), 4.36 (q, 1H), 3.90-3.80 (m, 3H), 3.52-3.27 (m,3H), 3.23 (t, 2H), 3.06-2.83 (m, 2H), 2.67-2.58 (m, 2H), 2.19 (s, 3H),2.09 (dp. 2H), 1.93 (d, 1H), 1.72-1.25 (m, 7H), 1.27-0.88 (m, 10H),0.88-0.70 (m, 13H). MS (ESI) m/e 1446.3 (M−H)⁻.

2.108 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[5-(1,3-benzothiazol-2-ylcarbamoyl)quinolin-3-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)-]dec-1-yl}oxy)ethyl](2-carboxyethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide(Synthon PW)

The title compound was prepared as described in Example 2.1, replacingExample 1.2.9 with Example 1.71. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆)δ ppm 9.97 (s, 1H), 9.70 (d, 1H), 9.40 (d, 1H), 8.31 (dd, 2H), 8.16 (d,1H), 8.05 (t, 2H), 8.01-7.91 (m, 2H), 7.78 (dd, 2H), 7.59 (d, 3H),7.52-7.44 (m, 2H), 7.36 (t, 1H), 7.26 (d, 2H), 6.96 (s, 2H), 5.99 (s,1H), 4.97 (s, 2H), 4.37 (d, 2H), 4.16 (dd, 1H), 3.53-3.20 (m, 9H), 2.94(dtd. 2H), 2.21 (s, 3H), 2.17-1.85 (m, 3H), 1.71-0.89 (m, 22H), 0.81(dd, 14H). MS (ESI) m/e 1410.4 (M−H)⁻.

2.109 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[1-(1,3-benzothiazol-2-ylcarbamoyl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide(Synthon QW)

The title compound was prepared by substituting Example 1.72.8 forExample 1.2.9 in Example 2.1. ¹H NMR (400 MHz, dimethyl sulfoxide d₆) δppm 11.07 (bs, 1H), 10.00 (bs, 1H), 8.27 (bs, 1H), 8.12 (m, 2H), 8.07(d, 1H), 7.99 (d, 1H), 7.84-7.74 (m, 2H), 7.65 (d, 1H), 7.59 (m, 2H),7.54-7.44 (m, 1H), 7.42-7.31 (m, 2H), 7.28 (m, 2H), 7.21 (q, 1H), 7.00(m, 1H) 6.94-6.92 (m, 2H), 6.04 (bs, 1H), 5.14 (s, 2H), 4.99 (m, 3H),4.39 (m, 2H), 4.30 (bs, 2H), 4.20 (m, 2H), 4.12 (bs, 2H), 3.70-3.64 (m,2H), 3.50 (m, 2H), 3.44-3.35 (m, 2H), 3.27 (m, 2H), 3.02 (m, 2H), 2.95(m, 2H), 2.68 (t, 2H), 2.14 (m, 4H), 1.96 (m, 1H), 1.69 (m, 1H), 1.58(m, 1H), 1.47 (m, 4H), 1.36 (m, 2H), 1.30-1.02 (m, 8H), 0.98 (m, 2H),0.85-0.80 (m, 16H),

2.110 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[7-(1,3-benzothiazol-2-ylcarbamoyl)-1H-indol-2-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-ornithinamide(Synthon RM)

Example 2.110 was prepared by substituting Example 1.74.6 for Example1.2.9 in Example 2.1. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm11.30 (s, 1H), 9.93 (s, 1H), 8.26 (d. 1H), 8.17 (d, 1H), 8.02 (d, 1H),7.92-7.84 (m, 3H), 7.76 (d, 1H), 7.69 (d, 1H), 7.54 (d, 3H), 7.47 (s,1H), 7.35 (dd, 2H), 7.22 (t, 3H), 7.08 (t, 1H), 6.93 (s, 2H), 4.90 (s,2H), 4.84 (t, 2H), 4.33 (q, 1H), 4.16-4.09 (m, I1H), 3.32 (t, 4H), 2.99(m, 6H), 2.21 (s, 3H), 2.09 (m, 2H), 1.91 (m, 1H), 1.71-0.71 (m, 25H).MS (ESI) m/e 1434.4 (M−H)⁻.

2.111 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-[4-({[{3-[8-(1,3-benzothiazol-2-ylcarbamoyl)-2-(6-carboxy-5-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}tricyclo[3.3.1.1^(3,7)]dec-1-y)methyl]-5-methyl-1H-pyrazol-4-yl}pyridin-2-yl)-1,2,3,4-tetrahydroisoquinolin-6-yl]propyl}(methyl)carbamoyl]oxy}methyl)phenyl]-N⁵-carbamoyl-L-ornithinamide(Synthon RR)

The title compound was prepared as described in Example 2.1, replacingExample 1.2.9 with Example 1.75.14. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 12.60 (bs, 1H), 9.98 (s, 1H), 8.33 (m, 2H), 8.02 (d,2H), 7.75 (d, 2H), 7.55 (d, 2H), 7.49 (m, 3H), 7.29 (m, 1H), 7.25 (s,4H), 6.99 (d, 2H), 6.95 (d, 1H), 5.90 (m, 1H), 5.42 (m, 2H), 4.95 (s,2H), 4.90 (m, 2H), 4.35 (t, 1). 4.18 (t, 1H), 3.85 (m, 2H), 3.80 (s,3H), 3.55 (s, 3H), 3.52 (m, 2H), 3.35 (m, 4H), 3.22 (m, 4H), 3.08 (m,2H), 2.99 (m, 2H), 2.92 (m, 2H), 2.85 (m, 2H), 2.79 (t, 2H), 2.52 (m,1H), 2.15 (m, 1H), 2.09 (s, 3H), 1.94 (m, 1H), 1.88 (m, 1H), 1.68 (m,1H), 1.54 (m, 1H), 1.42 (m, 4H), 1.38 (m, 4H), 1.27 (m, 4H), 1.13 (m,4H), 1.02 (m, 2H), 0.85 (s, 6H), 0.78 (m, 6H). MS (ESI) m/e 1523.3(M+H)⁺, 1521.6 (M−H)⁻

2.112 Synthesis ofN-(6-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}hexanoyl)-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-ornithinamide(Synthon SJ) 2.112.16-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-(2-((((4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)(2-sulfoethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinicAcid

Example 1.2.9, trifluoroacetic acid salt (390 mg), tert-butyl((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-1-oxobutan-2-yl)carbamate(286 mg) and 1-hydroxybenzotriazole hydrate (185 mg) inN,N-dimethylformamide (5 mL) was cooled in an ice-bath andN,N-diisopropylethylamine (0.35 mL) was added. The mixture was stirredat 0° C. for 30 minutes and at room temperature overnight. The reactionmixture was diluted with dimethyl sulfoxide to 10 mL and purified byreverse-phase HPLC on a Gilson system (C18 column), eluting with 20-80%acetonitrile in water containing 0.1% trifluoroacetic acid, to give thetitle compound. MS (ESI) m/e 680.1 (M+2H)²⁺.

2.112.23-(1-((3-(2-((((4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)(2-sulfoethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid

Example 2.112.1 (300 mg) in 10 mL of dichloromethane at 0° C. wastreated with trifluoroacetic acid (4 mL) for 30 minutes and the mixturewas concentrated. The residue was dissolved in a mixture of acetonitrileand water and lyophilized to provide the desired product as a TFA salt.MS (ESI) n/e 1257.4 (M−Hfy.

2.112.36-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-(2-((((4-((13S,16S)-13-isopropyl-2,2-dimethyl-4,11,14-trioxo-16-(3-ureidopropyl)-3-oxa-5,12,15-triazaheptadecanamido)benzyl)oxy)carbonyl)(2-sulfoethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinicAcid

Example 2.112.2 (trifluoroacetic acid salt, 385 mg,) and1-hydroxybenzotriazole hydrate (140 mg) in N,N-dimethylformamide (3 mL)was cooled in an ice-water bath. N,N-Diisopropylethylamine (226 μL) wasadded dropwise, followed by the addition of 2,5-dioxopyrrolidin-1-yl6-((tert-butoxycarbonyl)amino)hexanoate (127 mg), and the mixture wasstirred overnight. The mixture was purified by reverse-phase HPLC on aGilson system (C18 column), eluting with 20-75% acetonitrile in watercontaining 0.1% trifluoroacetic acid, to give the title compound. MS(ESI) m/e 1470.2 (M−H)⁻.

2.112.43-(1-((3-(2-((((4-((S)-2-((S)-2-(6-aminohexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)(2-sulfoethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid

The title compound was prepared using the procedure in Example 2.112.2,replacing

Example 2.112.1 with Example 2.112.3. MS (ESI) m/e 1370.5 (M−H)⁻.2.112.5N-(6-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}hexanoyl)-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.13,7]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N5-carbamoyl-L-ornithinamide

Example 2.112.4 (25 mg) and 2,5-dioxopyrrolidin-1-yl2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (9.19 mg) inN,N-dimethylformamide (0.3 mL) was treated withN,N-diisopropylethylamine (25.4 μL) for 30 minutes at 0° C. The reactionmixture was purified by reverse-phase HPLC on a Gilson system (C18column), eluting with 35-65% acetonitrile in 4 mM ammonium acetate watermixture, to provide the title compound as an ammonium salt. ¹H NMR (400MHz, dimethyl sulfoxide-d₆) δ 12.81 (s, 1H), 9.94 (s, 1H), 8.01 (dd,2H), 7.75 (d, 2H), 7.56 (s, 3H), 7.51-7.45 (m, 1H), 7.45-7.37 (m, 2H),7.36-7.28 (m, 2H), 7.24 (t, 3H), 7.17 (s, 2H), 7.05 (s, 3H), 7.04 (s,2H), 6.92 (s, 3H), 5.93 (s, 1H), 5.36 (s, 2H), 5.05-4.85 (m, 4H), 4.36(q, 1H), 4.16 (dd, 1H). 3.95 (s, 2H), 3.85 (t, 2H), 3.76 (d, 2H), 3.22(d, 1H), 3.05-2.81 (m, 6H), 2.68-2.53 (m, 2H), 2.09 (d, 4H), 1.76-0.86(m, 14H), 0.86-0.71 (m, 12H). MS (ESI) m/e 1507.5 (M−H)⁻.

2.113 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl][3-(beta-L-glucopyranuronosyloxy)propyl]carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide(Synthon SM)

The title compound was prepared as described in Example 2.1, replacingExample 1.2.9 with Example 1.87.3. ¹H NMR (501 MHz, dimethylsulfoxide-d₆) δ ppm 13.08 (s, 1H), 9.96 (s, 1H), 9.00 (s, 1H), 8.35 (dd,1H), 8.24-8.13 (m, 3H), 8.09-8.02 (m, 2H), 8.00 (d, 1H), 7.91 (d, 1H),7.77 (dd, 2H), 7.71-7.64 (m, 1H), 7.58 (t, 2H), 7.49-7.44 (m, 2H),7.39-7.32 (m.I H), 7.26 (d, 2H), 6.96 (s, 2H), 5.97 (s, 1H), 4.96 (s,2H), 4.37 (d, 1H), 4.22-4.12 (m, 2H), 3.84 (s, 1H), 3.37-3.20 (m, 6H),3.15 (t, 1H), 3.04-2.81 (m, 2H), 2.20 (s, 3H), 2.11 (dp. 2H), 1.99-1.88(m, 1H), 1.71 (q, 2H), 1.62-1.26 (m, 8H), 1.29-0.88 (m, 11H), 0.80 (dd,14H). MS (ESI) m/e 1571.4 (M−H)⁻.

2.114 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[4-(1,3-benzothiazol-2-ylcarbamoyl)isoquinolin-6-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide(Synthon SN)

The title compound was prepared as described in Example 2.1, replacingExample 1.2.9 with Example 1.78.5. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 9.95 (s, 1H), 9.61 (s, 1H), 9.08 (s, 1H), 9.00 (s,1H), 8.54 (dd, 1H), 8.43 (d, 1H), 8.24 (d, 1H), 8.08-7.95 (m, 3H), 7.77(dd, 2H), 7.63-7.51 (m, 2H), 7.50-7.42 (m, 2H), 7.40-7.31 (m, 1H), 7.24(d, 2H), 6.95 (s, 2H), 6.00 (s, 1H), 4.95 (d, 2H), 4.36 (q, 1H), 4.15(t, 1H), 3.27 (dt, 4H), 3.10-2.79 (m, 2H), 2.68-2.56 (m, 2H), 2.20 (s,3H), 1.98-1.84 (m, 1H), 1.72-0.87 (m, 19H), 0.79 (dd, 13H). MS (ESI) m/e1446.4 (M−H)⁻

2.115 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-alpha-glutamyl-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N-carbamoyl-L-ornithinamide(Synthon SS) 2.115.16-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-(2-((((4-((6S,9S,12S)-6-(3-(tert-butoxy)-3-oxopropyl)-9-isopropyl-2,2-dimethyl-4,7,10-trioxo-2-(3-ureidopropyl)-3-oxa-5,8,11-triazatridecanamido)benzyl)oxy)carbonyl)(2-sulfoethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinicAcid

To a mixture of Example 2.112.2 (85 mg), 1-hydroxybenzotriazole hydrate(41.3 mg), and (S)-5-tert-butyl 1-(2,5-dioxopyrrolidin-1-yl)2-((tert-butoxycarbonyl)amino)pentanedioate (54.0 mg) inN,N-dimethylformamide (3 mL) at 0° C. was addedN,N-diisopropylethylamine (118 μL) dropwise, and the mixture was stirredat 0° C. for 1 hour. The mixture was purified by reverse-phase HPLC on aGilson system (C18 column), eluting with 35-100% acetonitrile in watercontaining 0.1% trifluoroacetic acid, to give the title compound. MS(ESI) m/e 773.4 (M+2H)²⁺.

2.115.23-(1-((3-(2-((((4-((S)-2-((S)-2-((S)-2-amino-4-carboxybutanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)(2-sulfoethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid

Example 2.115.1 (100 mg) in dichloromethane (11 mL) at 0° C. was treatedwith trifluoroacetic acid (4 mL). The mixture was stirred at 0° C. for3.5 hours and concentrated. The residue was purified by reverse phaseHPLC, eluting with 5-60% acetonitrile in 0.1% trifluoroacetic acid watermixture to provide the title compound.

2.115.3N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-alpha-glutamyl-L-valyl-N-{4-[({[2-([3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl]-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.13,7]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N5-carbamoyl-L-ornithinamide

To a mixture of 1-hydroxybenzotriazole hydrate (2.87 mg).2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (5.77 mg) and Example2.115.2 (13 mg) at 0° C. was added N,N-diisopropylethylamine (13.08 sL),and the mixture was stirred at 0° C. for 1 hour. The reaction waspurified by reverse-phase HPLC on a Gilson system (C18 column), elutingwith 20-75% acetonitrile in water containing 0.1% trifluoroacetic acid,to give the title compound. ¹H NMR (501 MHz, dimethyl sulfoxide-d₆) δ12.83 (s, 1H), 9.99 (s, 1H), 8.13 (d, 1H), 8.02 (dd, 1H), 7.97 (d, 1H),7.80-7.74 (m, 1H), 7.64 (t, 1H), 7.61-7.48 (m, 4H), 7.47-7.38 (m, 2H),7.38-7.30 (m, 2H), 7.29-7.23 (m, 3H), 6.96 (s, 2H), 6.93 (d, 1H), 5.99(s, 1H), 5.06-4.88 (m, 5H), 4.37 (q, 1H), 4.28 (q, 1H), 4.18 (dd, 1H),3.86 (t, 2H), 3.78 (d, 2H), 3.34 (t, 3H), 3.23 (d, 2H), 2.99 (t, 3H),2.97-2.85 (m, 1H), 2.62 (dt, 1H), 2.26-2.15 (m, 2H), 2.16-2.00 (m, 5H),2.01-1.79 (m, 1H), 1.75-1.50 (m. 3H), 1.50-0.87 (m, 17H), 0.81 (dd,14H). MS (ESI) m/e 1579.6 (M−H)⁻.

2.116 Synthesis ofN-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L-alpha-glutamyl-L-valyl-N-({4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N-carbamoyl-L-ornithinamide(Synthon TA)

The title compound was prepared using the procedure in Example 2.115.3,replacing 2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate with2,5-dioxopyrrolidin-1-yl2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate. ¹H NMR (400 MHz,dimethyl sulfoxide-d₆) δ 10.02 (s, 1H), 8.38 (d, 1H), 8.14 (d, 1H), 8.03(d, 1H), 7.82 (dd, 2H), 7.60 (t, 3H), 7.55-7.40 (m, 3H), 7.35 (td, 2H),7.31-7.24 (m, 3H), 7.07 (s, 2H), 6.95 (d, 1H), 4.97 (d, 4H), 4.37 (ddd,2H), 4.23-4.05 (m, 3H), 3.88 (t, 6H), 3.80 (d, 2H), 3.25 (d, 2H),3.09-2.88 (m, 4H), 2.64 (s, 2H), 2.22 (dd. 2H), 2.09 (s, 3H), 2.02-1.49(m, 5H), 1.47-0.89 (m, 12H), 0.83 (dd, 12H). MS (ESI) m/e 1523.5 (M−H)⁻.

2.117 Synthesis of1-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]({[4-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-D-valyl-N⁵-carbamoyl-D-ornithyl}amino)benzyl]oxy}carbonyl)amino}-1,2-dideoxy-D-arabino-hexitol(Synthon TW)

The title compound was prepared by substituting Example 1.77.2 forExample 1.2.9 in Example 2.1. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δppm 12.85 (bs, 1H), 9.98 (s, 1H), 8.06 (d. 1H), 8.03 (d, 1H), 7.78 (t,2H), 7.60 (m, 3H), 7.52-7.42 (m, 4H), 7.36 (q, 2H), 7.28 (s, 1H), 7.27(d, 2H), 6.99 (s, 1H), 6.95 (d, 1H), 5.97 (bs, 1H), 5.00 (m, 2H), 4.95(s, 2H), 4.39 (m, 1H), 4.19 (m, 2H), 3.88 (t, 2H), 3.79 (m, 4H), 3.58(m, 4H), 3.46-3.33 (m, 10H), 3.26 (m, 4H), 3.01 (m, 2H), 2.94 (m. 1H),2.14 (m, 2H), 2.09 (s, 3H), 1.% (m, 1H), 1.69 (m, 2H), 1.59 (m, 1H),1.47 (m, 4H), 1.35 (m, 4H), 1.28-1.03 (m, 10H), 0.95 (m, 2H), 0.82 (m,12H). MS (ESI) m/e 1493 (M+H)⁺. 1491 (M−H)⁻.

2.118 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{(6-[4-(1,3-benzothiazol-2-ylcarbamoyl)-2-oxidoisoquinolin-6-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](methyl)carbamoyl}oxy)methyl]phenyl}-N-carbamoyl-L-ornithinamide(Synthon ST)

The title compound was prepared as described in Example 2.1, replacingExample 1.2.9 with Example 1.88.4. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 13.29 (s, 2H), 9.95 (s, 1H), 9.18 (s, 1H), 8.67 (s,1H), 8.57-8.36 (m, 1H), 8.29-7.87 (m, 4H), 7.77 (dd, 2H), 7.56 (d, 2H),7.53-7.41 (m, 2H), 7.24 (d, 2H), 6.95 (s, 2H), 5.95 (s, 1H), 4.94 (s,2H), 4.35 (q, 1H), 4.15 (dd, 1H), 3.84 (s, 3H), 3.28 (dt, 4H), 3.06-2.77(m, 3H), 2.19 (d, 3H), 2.17-1.80 (m, 3H), 1.74-0.88 (m, 22H), 0.79 (dd,13H). MS (ESI) m/e 1368.4 (M−H)⁻.

2.119 Synthesis ofN-({(3S,5S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-5-[(2-sulfoethoxy)methyl]pyrrolidin-1-yl}acetyl)-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide(Synthon ZL) 2.119.1(3R,7aS)-3-phenyltetrahydropyrrolo[1,2-c]oxazol-5(3H)-one

A mixture of (S)-5-(hydroxymethyl)pyrrolidin-2-one (25 g), benzaldehyde(25.5 g) and para-toluenesulfonic acid monohydrate (0.50 g) in toluene(300 mL) was heated to reflux using a Dean-Stark trap under a dryingtube for 16 hours. The reaction was cooled to room temperature, and thesolvent was decanted from the insoluble materials. The organic layer waswashed with saturated aqueous sodium bicarbonate mixture (2×) and brine(lx). The organic layer was dried over sodium sulfate, filtered andconcentrated under reduced pressure. The residue was purified by flashchromatography on silica gel, eluting with 35/65 heptane/ethyl acetate,to give the title compound. MS (DCI) mi/e 204.0 (M+H)+.

2.119.2(3R,6R,7aS)-6-bromo-3-phenyltetrahydropyrrolo[1,2-c]oxazol-5(3H)-one

To a cold (−77° C.) mixture of Example 2.119.1 (44.6 g) intetrahydrofuran (670 mL) was added lithium bis(trimethylsilyl)amide(1.0M in hexanes, 250 mL) dropwise over 40 minutes, keeping T_(rxn)<−73°C. The reaction was stirred at −77° C. for 2 hours, and bromine (12.5mL) was added dropwise over 20 minutes, keeping T_(rxn)<−64° C. Thereaction was stirred at −77° C. for 75 minutes and was quenched by theaddition of 150 mL cold I0% aqueous sodium thiosulfate mixture to the−77° C. reaction. The reaction was warmed to room temperature andpartitioned between half-saturated aqueous ammonium chloride mixture andethyl acetate. The layers were separated, and the organic layer waswashed with water and brine, dried over sodium sulfate, filtered andconcentrated under reduced pressure. The residue was purified by silicagel chromatography, eluting with a gradient of 80/20, 75/25, and 70/30heptane/ethyl acetate to give the title compound. MS (DCI) m/e 299.0 and301.0 (M+NH₃+H)⁺.

2.119.3(3R,6S,7aS)-6-bromo-3-phenyltetrahydropyrrolo[1,2-c]oxazol-5(3H)-one

The title compound was isolated as a by-product from Example 2.119.2. MS(DCI) m/e 299.0 and 301.0 (M+NH₃+H)⁺.

2.119.4(3R,6S,7aS)-6-azido-3-phenyltetrahydropyrro[1,2-c]oxazol-5(3H)-one

To a mixture of Example 2.119.2 (19.3 g) in N,N-dimethylformamide (100mL) was added sodium azide (13.5 g). The reaction was heated to 60° C.for 2.5 hours. The reaction was cooled to room temperature and quenchedby the addition of water (500 mL) and ethyl acetate (200 mL). The layerswere separated, and the organic layer was washed brine. The combinedaqueous layers were back-extracted with ethyl acetate (50 mL). Thecombined organic layers were dried with sodium sulfate, filtered andconcentrated under reduced pressure. The residue was purified by silicagel chromatography, eluting with 78/22 heptane/ethyl acetate, to givethe title compound. MS (DCI) m/e 262.0 (M+NH₃+H)⁺.

2.119.5(3R,6S,7aS)-6-amino-3-phenyltetrahydropyrrolo[1,2-c]oxazol-5(3H)-one

To a mixture of Example 2.119.4 (13.5 g) in tetrahydrofuran (500 mL) andwater (50 mL) was added polymer-supported triphenylphosphine (55 g). Thereaction was mechanically stirred overnight at room temperature. Thereaction was filtered through diatomaceous earth, eluting with ethylacetate and toluene. The mixture was concentrated under reducedpressure, dissolved in dichloromethane (100 mL), dried with sodiumsulfate, then filtered and concentrated to give the title compound,which was used in the subsequent step without further purification. MS(DCI) m/e 219.0 (M+H)⁺.

2.119.6(3R,6S,7aS)-6-(dibenzylamino)-3-phenyltetrahydropyrrolo[1,2-c]oxazol-5(3H)-one

To a mixture of Example 2.119.5 (11.3 g) in N,N-dimethylformamide (100mL) was added potassium carbonate (7.0 g), potassium iodide (4.2 g), andbenzyl bromide (14.5 mL). The reaction was stirred at room temperatureovernight and quenched by the addition of water and ethyl acetate. Thelayers were separated, and the organic layer was washed brine. Thecombined aqueous layers were back-extracted with ethyl acetate. Thecombined organic layers were dried with sodium sulfate, filtered andconcentrated under reduced pressure. The residue was purified by silicagel chromatography, eluting with a gradient of 10 to 15% ethyl acetatein heptane to give a solid that was triturated with heptane to give thetitle compound. MS (DCI) m/e 399.1 (M+H)⁺.

2.119.7 (3S,5S)-3-(dibenzylamino)-5-(hydroxymethyl)pyrrolidin-2-one

To a mixture of Example 2.119.6 (13 g) in tetrahydrofuran (130 mL) wasadded para-toluene sulfonic acid monohydrate (12.4 g) and water (50 mL),and the reaction was heated to 65° C. for 6 days. The reaction wascooled to room temperature and quenched by the addition of saturatedaqueous sodium bicarbonate and ethyl acetate. The layers were separated,and the organic layer was washed with brine. The combined aqueous layerswere back-extracted with ethyl acetate. The combined organic layers weredried with sodium sulfate, filtered and concentrated under reducedpressure. The waxy solids were triturated with heptane (150 mL) to givethe title compound. MS (DCI) m/e 311.1 (M+H)⁺.

2.119.8(3S,5S)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3-(dibenzylamino)pyrrolidin-2-one

To a mixture of Example 2.119.7 (9.3 g) and 1H-imidazole (2.2 g) inN,N-dimethylformamide was added tert-butylchlorodimethylsilane (11.2 mL,50 weight % in toluene), and the reaction mixture was stirred overnight.The reaction mixture was quenched by the addition of water and ethylether. The layers were separated, and the organic layer was washed withbrine. The combined aqueous layers were back-extracted with diethylether. The combined organic layers were dried with sodium sulfate,filtered and concentrated under reduced pressure. The residue waspurified by silica gel chromatography, eluting with 35% ethyl acetate inheptane, to give the title compound. MS (DCI) m/e 425.1 (M+H)⁺.

2.119.9 Tert-butyl2-((3S,5S)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3-(dibenzylamino)-2-oxopyrrolidin-1-yl)acetate

To a cold (0° C.) mixture of Example 2.119.8 (4.5 g) in tetrahydrofuran(45 mL) was added 95% sodium hydride (320 mg) in two portions. The coldmixture was stirred for 40 minutes, and tert-butyl 2-bromoacetate (3.2mL) was added. The reaction was warmed to room temperature and stirredovernight. The reaction was quenched by the addition of water and ethylacetate. The layers were separated, and the organic layer was washedwith brine. The combined aqueous layers were back-extracted with ethylacetate. The combined organic layers were dried with sodium sulfate,filtered and concentrated under reduced pressure. The residue waspurified by silica gel chromatography, eluting with a gradient of 5-12%ethyl acetate in heptane, to give the title compound. MS (DCI) m/e 539.2(M+H)⁺.

2.119.10 Tert-butyl2-((3S,5S)-3-(dibenzylamino)-5-(hydroxymethyl)-2-oxopyrrolidin-1-yl)acetate

To a mixture of Example 2.119.9 (5.3 g) in tetrahydrofuran (25 mL) wasadded tetrabutylammonium fluoride (11 mL, 1.0 M in 95/5tetrahydrofuran/water). The reaction was stirred at room temperature forone hour and then quenched by the addition of saturated aqueous ammoniumchloride mixture, water and ethyl acetate. The layers were separated,and the organic layer was washed with brine. The combined aqueous layerswere back-extracted with ethyl acetate. The combined organic layers weredried with sodium sulfate, filtered and concentrated under reducedpressure. The residue was purified by silica gel chromatography, elutingwith 35% ethyl acetate in heptane, to give the title compound. MS (DCI)m/e 425.1 (M+H)⁺.

2.119.11 Tert-buty2-((3S,5S)-5-((2-((4-((tert-butyldimethylsilyl)oxy)-2,2-dimethylbutoxy)sulfonyl)ethoxy)methyl)-3-(dibenzylamino)-2-oxopyrrolidin-1-yl)acetate

To a mixture of Example 2.119.10 (4.7 g) in dimethyl sulfoxide (14 mL)was added a mixture of4-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylbutyl ethenesulfonate (14.5g) in dimethyl sulfoxide (14 mL). Potassium carbonate (2.6 g) and water(28 μL) were added, and the reaction was heated at 60° C. under nitrogenfor one day. The reaction was cooled to room temperature, and thenquenched by the addition of brine mixture, water and diethyl ether. Thelayers were separated, and the organic layer was washed with brine. Thecombined aqueous layers were back-extracted with diethyl ether. Thecombined organic layers were dried with sodium sulfate, filtered andconcentrated under reduced pressure. The residue was purified by silicagel chromatography, eluting with a gradient of 15-25% ethyl acetate inheptane, to give the title compound. MS (ESI+) m/e 871.2 (M+H)+.

2.119.12 Tert-butyl2-((3S,5S)-3-amino-5-((2-((4-((tert-butyldimethylsilyl)oxy)-2,2-dimethylbutoxy)sulfonyl)ethoxy)methyl)-2-oxopyrrolidin-1-yl)acetate

Example 2.119.11 (873 mg) was dissolved in ethyl acetate (5 mL) andmethanol (15 mL), and palladium hydroxide on carbon, 20% by wt (180 mg)was added. The reaction mixture was stirred under a hydrogen atmosphere(30 psi) at room temperature for 30 hours, then at 50° C. for one hour.The reaction was cooled to room temperature, filtered, and concentratedto give the desired product. MS (ESI+) m/e 691.0 (M+H)⁺.

2.119.134-(((3S,5S)-1-(2-(tert-butoxy)-2-oxoethyl)-5-((2-((4-((tert-butyldimethylsilyl)oxy)-2,2-dimethylbutoxy)sulfonyl)ethoxy)methyl)-2-oxopyrrolidin-3-yl)amino)-4-oxobut-2-enoicAcid

Maleic anhydride (100 mg) was dissolved in dichloromethane (0.90 mL),and a mixture of Example 2.119.12 (650 mg) in dichloromethane (0.90 mL)was added dropwise, then heated at 40° C. for 2 hours. The reactionmixture was directly purified by silica gel chromatography, eluting witha gradient of 1.0-2.5% methanol in dichloromethane containing 0.2%acetic acid. After concentrating the product-bearing fractions, toluene(10 mL) was added, and the mixture was concentrated again to give thetitle compound. MS (ESI-) m/e 787.3 (M−H)⁻.

2.119.14 Tert-butyl2-((3S,5S)-5-((2-((4-((tert-butyldimethylsilyl)oxy)-2,2-dimethylbutoxy)sulfonyl)ethoxy)methyl)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxopyrrolidin-1-yl)acetate

Example 2.119.13 (560 mg) was slurried in toluene (7 mL), andtriethylamine (220 μL) and sodium sulfate (525 mg) were added. Thereaction was heated at reflux under a nitrogen atmosphere for 6 hours,and the reaction mixture was stirred at room temperature overnight. Thereaction was filtered, and the solids were rinsed with ethyl acetate.The eluent was concentrated under reduced pressure, and the residue waspurified by silica gel chromatography, eluting with 45/55 heptane/ethylacetate to give the title compound.

2.119.152-((3S,5S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-5-((2-sulfoethoxy)methyl)pyrrolidin-1-yl)aceticAcid

Example 2.119.14 (1.2 g) was dissolved in trifluoroacetic acid (15 mL)and heated to 65-70° C. under nitrogen overnight. The trifluoroaceticacid was removed under reduced pressure. The residue was dissolved inacetonitrile (2.5 mL) and purified by preparative reverse-phase liquidchromatography on a Luna C18(2) AXIA column (250×50 mm, 10 μm particlesize) using a gradient of 5-75% acetonitrile containing 0.1%trifluoroacetic acid in water over 30 minutes, to give the titlecompound. MS (ESI-) m/e 375.2 (M−H)⁻.

2.119.163-(1-((3-(2-((((4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)(2-sulfoethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)naphthaen-2-yl)picolinicAcid

The title compound was prepared by substituting Example 1.43.7 forExample 1.2.9 in Example 2.49.1. MS (ESI-) m/e 1252.4 (M−H)⁻.

2.119.17N-({(3S,5S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-5-[(2-sulfoethoxy)methyl]pyrrolidin-1-yl}acetyl)-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.13,7]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N5-carbamoyl-L-ornithinamide

Example 2.119.15 (7 mg) was dissolved in N,N-dimethylformamide (0.15mL), and O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (9 mg) and N,N-diisopropylethylamine (7 μL) wereadded. The mixture was stirred for 3 minutes at room temperature andadded to a mixture of Example 2.119.16 (28 mg) andN,N-diisopropylethylamine (15 μL) in N,N-dimethylformamide (0.15 mL).After 1 hour, the reaction was diluted with N,N-dimethylformamide/water1/1 (1.0 mL) and purified by reverse-phase chromatography (C18 column),eluting with 5-75% acetonitrile in 0.1% TFA water, to provide the titlecompound. ¹H NMR (500 MHz, dimethyl sulfoxide-d₆) δ ppm 9.95 (s, 1H),9.02 (s, 1H), 8.37 (d, 1H), 8.22 (m 2H), 8.18 (m, 2H), 8.08 (m, 2H),8.03 (m, 1H), 7.96 (br d, 1H), 7.81 (d, 1H), 7.70 (t, 1H), 7.61 (br m,3H), 7.48 (m, 2H), 7.37 (t, 1H), 7.27 (br m, 2H), 7.08 (s, 2H), 4.99 (brd, 3H), 4.68 (t, 1H), 4.39 (m, 1H), 4.20 (m, 2H), 4.04 (m, 1H), 3.87 (brd, 2H), 3.74 (br m, 1H) 3.65 (br t, 2H), 3.48 (br m, 4H), 3.43 (br m,2H), 3.26 (br m, 2H), 3.00 (br m, 2H), 2.80 (m, 1H), 2.76 (m, 1H), 2.66(br m, 2H), 2.36 (br m, 1H), 2.22 (s, 3H), 2.00 (m, 1H), 1.87 (m, 1H),1.69 (br m, 1H), 1.62 (br m, 1H), 1.40 (br m, 4H), 1.31-1.02 (m, 10H),0.96 (m, 2H), 0.85 (m, 12H). MS (ESI−) m/e 1610.3 (M−H)⁻.

2.120 Synthesis ofN-{(2S)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-[4-(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yloxy)phenyl]propanoyl}-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide(Synthon SX) 2.120.1 (S)-methyl3-(4-(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yloxy)phenyl)-2-((tert-butoxycarbonyl)amino)propanoate

To a mixture of2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-v4-methylbenzenesulfonate(82.48 g) and potassium carbonate (84.97 g) in acetonitrile (1.5 L) wasadded (S)-methyl2-((tert-butoxycarbonyl)amino)-3-(4-hydroxyphenyl)propanoate (72.63 g),and the reaction mixture was stirred at 30° C. for 12 hours. After LC/MSindicated the starting material was consumed and the major product wasthe desired product, the reaction was filtered, and the filtrate wasconcentrated to afford the crude product which was purified by prep-HPLCto provide the title compound. MS (ESI): m/e 811 (M+H₂O)⁺.

2.120.23-(4-(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yloxy)phenyl)-2-((tert-butoxycarbonyl)amino)propanoicAcid

To a mixture of Example 2.120.1 (90.00 g) in tetrahydrofuran (1.5 L) andwater (500 mL) was added lithium hydroxide monohydrate (14.27 g). Thereaction mixture was stirred at 30° C. for 12 hours, and LC/MS indicatedthe starting material was consumed and the major product was the desiredproduct. The reaction mixture was adjusted using aqueous HCl to pH=6,and the mixture was concentrated to provide the crude title compound. MS(ESI): m/e 778.3 (M−H)⁻.

2.120.33-(4-(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yloxy)phenyl)-2-aminopropanoicAcid

To a mixture of Example 2.120.2 (88.41 g) in dichloromethane (1.5 L) wasadded trifluoroacetic acid (100 mL) at 25° C. under N₂, and the reactionmixture was stirred at 40° C. for 12 hours. LC/MS indicated the startingmaterial was consumed, and the major product was the desired product.The mixture was concentrated to afford the crude product which waspurified by prep-HPLC provide the title compound as a trifluoroaceticacid salt. ¹H NMR (400 MHz, CDCl₃) δ ppm 7.20 (d, J=8.6 Hz, 2H), 6.93(d, J=8.2 Hz, 2H), 4.22 (dd, J=5.5, 7.4 Hz, 1H), 4.14-4.06 (m, 2H),3.84-3.79 (m, 2H), 3.68-3.50 (m, 40H), 3.33 (s, 3H), 3.21 (d. J=5.5 Hz.1H), 3.12-3.05 (m, 1H). MS (ESI) m/e 680.1 (M+H)+.

2.120.44-((2-(4-(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yloxy)phenyl)-1-carboxyethyl)amino)-4-oxobut-2-enoicAcid

To a mixture of Example 2.120.3 (80.00 g) in dioxane (1 L) was addedfuran-2, 5-dione (35 g), and the reaction mixture was stirred at 120° C.for 4 hours. LC/MS indicated the starting material was consumed, and themajor product was the desired product. The mixture was concentrated toafford crude title compound which was used without purification in thenext step. MS (ESI) m/e 795.4 (M+H)⁺.

2.120.5(S)-3-(4-(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yloxy)phenyl)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoicAcid

To a mixture of Example 2.120.4 (96 g, crude) in toluene (1.5 L) and wasadded triethylamine (35.13 g), and the reaction mixture was stirred at120° C. for 4 hours. LC/MS indicated the starting material was consumed,and the major product was the desired product. The reaction was filteredto isolate the organic phase, and the organics were concentrated toafford the crude product which was purified by prep-HPLC (Instrument:Shimadzu LC-20AP preparative HPLC. Column: Phenomenex® Luna® (2) C18250*50 mm i.d. 10u, Mobile phase: A for H₂O (0.09% trifluoroacetic acid)and B for CH₃CN, Gradient: B from 15% to 43% in 20 minutes, Flow rate:80 ml/minute, Wavelength: 220 & 254 nm, Injection amount: 1 gram perinjection), followed by SFC-HPLC to provide the title compound. ¹H NMR(400 MHz, CDCl₃) δ ppm 6.98 (d, 2H), 6.74 (d, 2H), 6.56 (s, 2H), 4.85(dd, 1H), 4.03 (t, 2H), 3.84-3.76 (m, 2H), 3.71-3.66 (m, 2H), 3.65-3.58(m, 39H), 3.55-3.50 (m 2H), 3.41-3.30 (m, 4H). MS (ESI) m/e 760.3(M+H)⁺.

2.120.6N-{(2S)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-[⁴-(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yloxy)phenyl]propanyl}-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.13,7]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N5-carbamoyl-L-ornithinamide

The title compound was prepared by substituting Example 2.120.5 forExample 2.119.15 in Example 2.119.17. ¹H NMR (400 MHz dimethylsulfoxide-d₆) δ ppm 10.03 (s, 1H), 9.02 (s, 1H), 8.37 (d, 1H), 8.22 (m,3H), 8.16 (d, 1H), 8.12 (br m, 1H), 8.07 (d, 1H), 8.01 (d, 1H), 7.96 (brd, 1H), 7.81 (d, 1H), 7.70 (t, 1H), 7.59 (br m, 2H), 7.48 (m, 2H), 7.37(t, 1H), 7.28 (d, 2H), 7.02 (d, 2H), 6.89 (s, 2H), 6.77 (d, 2H), 4.98(br d. 2H), 4.79 (dd, 1H), 4.39 (br m, 1H), 4.23 (br m, 2H), 3.99 (br m,2H), 3.88 (br m, 2H), 3.69 (br m, 4H), 3.55 (m, 4H), 3.50 (s, 32H), 3.42(m, 4H), 3.27 (m, 4H), 3.23 (s, 3H), 3.20 (m, 1H), 3.03 (br m, 1H), 2.98(m, 1H), 2.65 (br t, 2H), 2.22 (s, 3H), 1.97 (br m, 1H), 1.69 (br m,1H), 1.61 (br m, 1H), 1.39 (m, 4H), 1.31-0.91 (m, 12H), 0.85 (m, 9H),0.77 (d, 3H). MS (ESI) m/e 1993.7 (M−H)⁻.

2.121 Synthesis ofN-({(3S,5S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-5-[(2-sulfoethoxy)methyl]pyrrolidin-1-yl}acetyl)-L-valyl-N-{(4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide(Synthon SW)

The title compound was prepared by substituting Example 2.49.1 forExample 2.119.16 in Example 2.119.17. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 9.96 (s, 1H), 8.17 (br d, 1H), 8.03 (d, 2H), 7.79(d, 1H), 7.61 (m, 3H), 7.55 (d, 1H), 7.45 (m, 2H), 7.37 (m, 3H), 7.27(d, 2H), 7.08 (s, 2H), 6.98 (d, 1H), 4.97 (m, 4H), 4.68 (t, 1H), 4.37(br m, 1H), 4.22 (br s, 1H), 4.17 (d, 1H), 4.03 (d, 1H), 3.89 (br t,2H), 3.83 (br d, 2H), 3.74 (br m, 1H), 3.65 (t, 2H), 3.49 (m, 3H), 3.40(br m, 4H), 3.25 (br m, 2H), 3.02 (br m, 4H), 2.80 (m, 2H), 2.67 (br m,2H), 2.37 (br m, 1H), 2.10 (s, 3H), 1.99 (m, 1H), 1.86 (m, 1H), 1.69 (brm, 1H), 1.61 (br m, 1H), 1.52-0.91 (m, 16H), 0.85 (m, 12H). MS (ESI) m/e1615.4 (M−H)⁻.

2.122 Synthesis ofN-{(2S)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-[4-(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yloxy)phenyl]propanoyl}-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N-carbamoyl-L-ornithinamide(Synthon TV)

To a mixture of Example 2.120.5 (19.61 mg), and-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (9.81 mg) in N,N-dimethylformamide (0.8 mL) wasadded N,N-diisopropylethylamine (27.7 μL). The mixture was stirred for 5minutes and added to a cold mixture of Example 2.112.2 inN,N-dimethylformamide (0.5 mL) at 0° C. The reaction mixture was stirredat 0° C. for 40 minutes, and purified by reverse-phase HPLC on a Gilsonsystem (C18 column), eluting with 20-80% acetonitrile in watercontaining 0.1% trifluoroacetic acid, to give the title compound. ¹H NMR(400 MHz, dimethyl sulfoxide-d₆) δ 9.99 (s, 1H), 8.19 (d, 1H), 8.14-8.04(m. 1H), 8.00 (dd, 1H), 7.75 (d, 1H), 7.62-7.52 (m, 3H), 7.49 (d, 1H),7.46-7.37 (m, 2H), 7.36-7.29 (m, 2H), 7.28-7.21 (m, 3H), 6.99 (d, 2H),6.92 (d, 1H), 6.85 (s, 2H), 6.79-6.71 (m, 2H), 4.94 (d, 3H), 4.76 (dd,1H), 4.35 (d, 1H), 4.20 (t, 1H), 3.% (dd, 2H), 3.85 (t, 2H), 3.77 (d,2H), 3.66 (dd, 2H), 3.52 (dd, 2H), 3.50-3.47 (m, 2H), 3.39 (dd, 2H),3.20 (s, 4H), 2.97 (t, 3H), 2.60 (t, 2H), 2.13-2.01 (m, 3H), 1.93 (s,1H), 1.61 (d, 2H), 1.49-0.88 (m, 10H), 0.87-0.59 (m, 12H). MS (ESI) m/e1998.7 (M−H)⁻.

2.123 Synthesis of(6S)-2,6-anhydro-6-(2-{2-[([2-(3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl)oxy)methyl]-5-({N-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L-valyl-L-alanyl}amino)phenyl)ethyl)-L-gulonicacid (Synthon SZ) 2.123.1(3R,4S,5R,6R)-3,4,5-tris(benzyloxy)-6-(benzyloxymethyl)-tetrahydropyran-2-one

To a mixture of(3R,4S,5R,6R)-3,4,5-tris(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-2-ol(75 g) in dimethyl sulfoxide (400 mL) at 0° C. was added aceticanhydride (225 mL). The mixture was stirred for 16 hours at roomtemperature before it was cooled to 0° C. A large volume of water wasadded, and stirring was stopped so that the reaction mixture was allowedto settle for 3 hours (the crude lactone migrated to the bottom of theflask). The supernatant was removed, and the crude mixture was dilutedwith ethyl acetate and was washed 3 times with water, neutralized withsaturated aqueous mixture of NaHCO₃, and washed again twice with water.The organic layer was then dried over magnesium sulfate, filtered andconcentrated to give the title compound. MS (ESI) m/e 561 (M+Na)⁺.

2.123.2(3R,4S,5R,6R)-3,4,5-tris(benzyloxy)-6-(benzyloxymethyl)-2-ethynyl-tetrahydro-2H-pyran-2-ol

To a mixture of ethynyltrimethylsilane (18.23 g) in tetrahydrofuran (400mL) under nitrogen and chilled in a dry ice/acetone bath (internal temp−65° C.) was added 2.5M BuLi in hexane (55.7 mL) dropwise, keeping thetemperature below −60° C. The mixture was stirred in a cold bath for 40minutes, followed by an ice-water bath (internal temp rose to 0.4° C.)for 40 minutes, and finally cooled to −75° C., again. A mixture ofExample 2.123.1 (50 g) in tetrahydrofuran (50 mL) was added dropwise,keeping the internal temperature below −70° C. The mixture was stirredin a dry ice/acetone bath for additional 3 hours. The reaction wasquenched with saturated aqueous NaHCO₃ mixture (250 mL). The mixture wasallowed to warm to room temperature, extracted with ethyl acetate (3×300mL), dried over MgSO₄, filtered, and concentrated in vacuo to give thetitle compound. MS (ESI) m/e 659 (M+Na)⁺.

2.123.3trimethyl(((3S,4R,5R,6R)-3,4,5-tris(benzyloxy)-6-(benzyloxymethyl)-tetrahydro-2H-pyran-2-yl)ethynyl)silane

To a mixed mixture of Example 2.123.2 (60 g) in acetonitrile (450 mL)and dichloromethane (150 mL) at −15° C. in an ice-salt bath was addedtriethylsilane (81 mL) dropwise, followed by addition of borontrifluoride diethyl ether complex (40.6 mL) at such a rate that theinternal temperature did not exceed −10° C. The mixture was then stirredat −15° C. to −10° C. for 2 hours. The reaction was quenched withsaturated aqueous NaHCO₃ mixture (275 mL) and stirred for 1 hour at roomtemperature. The mixture was then extracted with ethyl acetate (3×550mL). The extracts were dried over MgSO₄, filtered, and concentrated. Theresidue was purified by flash chromatography eluting with a gradient of0% to 7% ethyl acetate/petroleum ether to give the title compound. MS(ESI) m/e 643 (M+Na)⁺.

2.123.4(2R,3R,4R,5S)-3,4,5-tris(benzyloxy)-2-(benzyloxymethyl)-6-ethynyl-tetrahydro-2H-pyran

To a mixed mixture of Example 2.123.3 (80 g) in dichloromethane (200 mL)and methanol (1000 mL) was added 1N aqueous NaOH mixture (258 mL). Themixture was stirred at room temperature for 2 hours. The solvent wasremoved. The residue was then partitioned between water anddichloromethane. The extracts were washed with brine, dried over Na₂SO₄,filtered, and concentrated to give the title compound. MS (ESI) m/e 571(M+Na)⁺.

2.123.5(2R,3R,4R,5S)-2-(acetoxymethyl)-6-ethynyl-tetrahydro-2H-pyran-3,4,5-triylTriacetate

To a mixture of Example 2.123.4 (66 g) in acetic anhydride (500 mL)cooled by an ice/water bath was added boron trifluoride diethyl ethercomplex (152 mL) dropwise. The mixture was stirred at room temperaturefor 16 hours, cooled with an ice/water bath and neutralized withsaturated aqueous NaHCO₃ mixture. The mixture was extracted with ethylacetate (3×500 mL), dried over Na₂SO₄, filtered, and concentrated invacuo. The residue was purified by flash chromatography eluting with agradient of 0% to 30% ethyl acetate/petroleum ether to give the titlecompound. MS (ESI) m/e 357 (M+H)⁺.

2.123.6(3R,4R,5S,6R)-2-ethynyl-6-(hydroxymethyl)-tetrahydro-2H-pyran-3,4,5-triol

To a mixture of Example 2.123.5 (25 g) in methanol (440 mL) was addedsodium methanolate (2.1 g). The mixture was stirred at room temperaturefor 2 hours, and then neutralized with 4 M HCl in dioxane. The solventwas removed, and the residue was adsorbed onto silica gel and loadedonto a silica gel column. The column was eluted with a gradient of 0 to100% ethyl acetate/petroleum ether then 0% to 12% methanol/ethyl acetateto give the title compound. MS (ESI) m/e 211 (M+Na)⁺.

2.123.7(2S,3S,4R,5R)-6-ethynyl-3,4,5-trihydroxy-tetrahydro-2H-pyran-2-carboxylicAcid

A three-necked round bottom flask was charged with Example 2.123.6 (6.00g). KBr (0.30 g), tetrabutylammonium bromide (0.41 g) and 60 mL ofsaturated aqueous NaHCO₃ mixture. TEMPO((2,2,6,6-tetramethylpiperidin-1-yl)oxyl, 0.15 g) in 60 mLdichloromethane was added. The mixture was stirred vigorously and cooledin an ice-salt bath to −2° C. internal temperature. A mixture of brine(12 mL), aqueous NaHCO₃ mixture (24 mL) and NaOCI (154 mL) was addeddropwise such that the internal temperature was maintained below 2° C.The pH of the reaction mixture was maintained in the 8.2-8.4 range withthe addition of solid Na2CO₃. After a total of 6 hours, the reactionmixture was cooled to 3° C. internal temperature and ethanol (˜20 mL)was added dropwise. The mixture was stirred for ˜30 minutes. The mixturewas transferred to a separatory funnel, and the dichloromethane layerwas discarded. The pH of the aqueous layer was adjusted to 2-3 using 1 Maqueous HCl. The aqueous layer was then concentrated to dryness toafford a solid. Methanol (100 mL was) added to the dry solid, and theslurry was stirred for ˜30 minutes. The mixture was filtered over a padof diatomaceous earth, and the residue in the funnel was washed with˜100 mL of methanol. The filtrate was concentrated under reducedpressure to obtain the title compound.

2.123.8 (2S,3S,4R,5R)-methyl6-ethynyl-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylate

A 500 mL three-necked round bottom flask was charged with a suspensionof Example 2.123.7 (6.45 g) in methanol (96 mL) and was cooled in anice-salt-bath with internal temperature of −1° C. Neat thionyl chloride(2.79 mL) was carefully added. The internal temperature kept risingthroughout the addition but did not exceed 10° C. The reaction wasallowed to slowly warm up to 15-20° C. over 2.5 hours. After 2.5 hours,the reaction was concentrated to give the title compound.

2.123.9(3S,4R,5S,6S)-2-ethynyl-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate

To Example 2.123.8 (6.9 g) as a mixture in N,N-dimethylformamide (75 mL)was added 4-(dimethylamino)pyridine (0.17 g) and acetic anhydride (36.1mL). The suspension was cooled in an ice-bath and pyridine (18.04 mL)was added via syringe over 15 minutes. The reaction was allowed to warmto room temperature overnight. Additional acetic anhydride (12 mL) andpyridine (6 mL) were added and stirring was continued for an additional6 hours. The reaction was cooled in an ice-bath and 250 mL of saturatedaqueous NaHCO₃ mixture was added and stirred for 1 hour. Water (100 mL)was added, and the mixture was extracted with ethyl acetate. The organicextract was washed twice with saturated CuSO₄ mixture, dried, filtered,and concentrated. The residue was purified by flash chromatography,eluting with 50% ethyl acetate/petroleum ether to give the titlecompound. ¹H NMR (500 MHz, methanol-d) δ ppm 5.29 (t, 1H), 5.08 (td,2H), 4.48 (dd, 1H) 4.23 (d, 1H), 3.71 (s, 3H), 3.04 (d, 1H), 2.03 (s,3H), 1.99 (s, 3H), 1.98 (s, 4H),

2.123.10 2-iodo-4-nitrobenzoic Acid

A 3L fully jacketed flask equipped with a mechanical stirrer,temperature probe and an addition funnel under a nitrogen atmosphere,was charged with 2-amino-4-nitrobenzoic acid (69.1 g. Combi-Blocks) andsulfuric acid, 1.5 M aqueous (696 mL). The resulting suspension wascooled to 0° C. internal temperature, and a mixture of sodium nitrite(28.8 g) in water (250 mL) was added dropwise over 43 minutes with thetemperature kept below 1° C. The reaction was stirred at ca. 0° C. for 1hour. A mixture of potassium iodide (107 g) in water (250 mL) was addeddropwise over 44 minutes with the internal temperature kept below 1° C.(Initially addition was exothermic and there was gas evolution). Thereaction was stirred 1 hour at 0° C. The temperature was raised to 20°C., and then stirred at ambient temperature overnight. The reactionmixture became a suspension. The reaction mixture was filtered, and thecollected solid was washed with water. The wet solid (108 g) was stirredin 10% sodium sulfite (350 ml, with ˜200 mL water used to wash in thesolid) for 30 minutes. The suspension was acidified with concentratedhydrochloric acid (35 mL), and the solid was collected by filtration andwashed with water. The solid was slurried in water (1L) and re-filtered,and the solid was left to dry in the funnel overnight. The solid wasthen dried in a vacuum oven for 2 hours at 60° C. The resulting solidwas triturated with dichloromethane (500 mL), and the suspension wasfiltered and washed with additional dichloromethane. The solid wasair-dried to give the title compound

2.123.11 (2-iodo-4-nitrophenyl)methanol

A flame-dried 3 L 3-necked flask was charged with Example 2.123.10 (51.9g) and tetrahydrofuran (700 mL). The mixture was cooled in an ice bathto 0.5° C., and borane-tetrahydrofuran complex (443 mL, 1M in THF) wasadded dropwise (gas evolution) over 50 minutes, reaching a finalinternal temperature of 1.3° C. The reaction mixture was stirred for 15minutes, and the ice bath was removed. The reaction was left to come toambient temperature over 30 minutes. A heating mantle was installed, andthe reaction was heated to an internal temperature of 65.5° C. for 3hours, and then allowed to cool to room temperature while stirringovernight. The reaction mixture was cooled in an ice bath to 0° C., andquenched by dropwise addition of methanol (400 mL). After a briefincubation period, the temperature rose quickly to 2.5° C. with gasevolution. After the first 100 mL are added over ˜30 minutes, theaddition was no longer exothermic, and the gas evolution ceased. The icebath was removed, and the mixture was stirred at ambient temperatureunder nitrogen overnight. The mixture was concentrated to a solid,dissolved in dichloromethanemethanol and adsorbed on to silica gel (˜150g). The residue was loaded on a plug of silica gel (3000 mL) and elutedwith dichloromethane to give the title compound.

2.123.12 (4-amino-2-iodophenyl)methanol

A 5 L flask equipped with a mechanical stirrer, heating mantlecontrolled by a JKEM temperature probe and a condenser was charged withExample 2.123.11 (98.83 g) and ethanol (2 L). The reaction was stirredrapidly, and iron (99 g) was added, followed by a mixture of ammoniumchloride (20.84 g) in water (500 mL). The reaction was heated over thecourse of 20 minutes to an internal temperature of 80.3° C., where itbegan to reflux vigorously. The mantle was dropped until the refluxcalmed. Thereafter, the mixture was heated to 80° C. for 1.5 hour. Thereaction was filtered hot through a membrane filter, and the ironresidue was washed with hot 50% ethyl acetate/methanol (800 mL). Theeluent was passed through a diatomaceous earth pad, and the filtrate wasconcentrated. The residue was partitioned between 50% brine (1500 mL)and ethyl acetate (1500 mL). The layers were separated, and the aqueouslayer was extracted with ethyl acetate (400 mL×3). The combined organiclayers were dried over sodium sulfate, filtered and concentrated to givethe title compound, which was used without further purification.

2.123.13 4-(((tert-butyldimethylsilyl)oxy)methyl)-3-iodoaniline

A 5 L flask with a mechanical stirrer was charged with Example 2.123.12(88 g) and dichloromethane (2 L). The suspension was cooled in an icebath to an internal temperature of 2.5° C., andtert-butylchlorodimethylsilane (53.3 g) was added portion-wise over 8minutes. After 10 minutes, 1H-imidazole (33.7 g) was added portionwiseto the cold reaction. The reaction was stirred 90 minutes while theinternal temperature rose to 15° C. The reaction mixture was dilutedwith water (3 L) and dichloromethane (1 L). The layers were separated,and the organic layer was dried over sodium sulfate, filtered, andconcentrated to an oil. The residue was purified by silica gelchromatography (1600 g silica gel), eluting a gradient of 0-25% ethylacetate in heptane, to give the title compound as an oil.

2.123.14(S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanoicAcid

To a mixture of(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanoic acid(6.5 g) in dimethoxyethane (40 mL) was added (S)-2-aminopropanoic acid(1.393 g) and sodium bicarbonate (1.314 g) in water (40 mL).Tetrahydrofuran (20 mL) was added to aid solubility. The resultingmixture was stirred at room temperature for 16 hours. Aqueous citricacid (15%. 75 mL) was added, and the mixture was extracted with 10%2-propanol in ethyl acetate (2×100 mL). A precipitate formed in theorganic layer. The combined organic layers were washed with water (2×150mL). The organic layer was concentrated under reduced pressure and thentriturated with diethyl ether (80 mL). After brief sonication, the titlecompound was collected by filtration. MS (ESI) m/e 411 (M+H)⁺.

2.123.15 (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-(((tert-butyldimethylsilyl)oxy)methyl)-3-iodophenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate

A mixture of Example 2.123.13 (5.44 g) and Example 2.123.14 (6.15 g) ina mixture of dichloromethane (70 mL) and methanol (35.0 mL) was addedethyl 2-ethoxyquinoline-1(2H)-carboxylate (4.08 g), and the reaction wasstirred overnight. The reaction mixture was concentrated and loaded ontosilica gel, eluting with a gradient of 10% to 95% heptane in ethylacetate followed by 5% methanol in dichloromethane. Theproduct-containing fractions were concentrated, dissolved in 0.2%methanol in dichloromethane (50 mL), loaded onto silica gel and elutedwith a gradient of 0.2% 10 to 2% methanol in dichloromethane. Theproduct containing fractions were collected to give the title compound.MS (ESI) m/e 756.0 (M+H)⁺.

2.123.16(2S,3S,4R,5S,6S)-2-((5-((S)-2-((S)-2-((((9H-fluoren-9-y)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)-2-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)ethynyl)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate

A mixture of Example 2.123.9 (4.500 g). Example 2.123.15 (6.62 g),copper( ) iodide (0.083 g) and bis(triphenylphosphine)palladium(II)dichloride (0.308 g) were combined in vial and degassed.N,N-dimethylformamide (45 mL) and N-ethyl-N-isopropylpropan-2-amine(4.55 mL) were added, and the reaction vessel was flushed with nitrogenand stirred at room temperature overnight.

The reaction was partitioned between water (100 mL) and ethyl acetate(250 mL). The layers were separated, and the organic layer was driedover magnesium sulfate, filtered, and concentrated. The residue waspurified by silica gel chromatography, eluting with a gradient of 5% to95% ethyl acetate in heptane. The product containing fractions werecollected, concentrated and purified by silica gel chromatography,eluting with a gradient of 0.25% to 2.5% methanol in dichloromethane togive the title compound. MS (ESI) m/e 970.4 (M+H)⁺.

2.123.17(2S,3S,4R,5S,6S)-2-(5-((S)-2-((S)-2-((((9H-fluoren-9-y)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)-2-(((tert-butyldimethylsilyl)oxy)methyl)phenethyl)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,45-triylTriacetate

Example 2.123.16 (4.7 g) and tetrahydrofuran (95 mL) were added to 5%Pt/C (2.42 g, wet) in a 50 mL pressure bottle and shaken for 90 minutesat room temperature under 50 psi of hydrogen. The reaction was filteredand concentrated to give the title compound. MS (ESI) m/e 974.6 (M+H)⁺.

2.123.18(2S,3S,4R,5S,6S)-2-(5-((S)-2-((S)-2-((((9H-fluoren-9-y)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)-2-(hydroxymethyl)phenethyl)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate

A mixture of Example 2.123.17 (5.4 g) in tetrahydrofuran (7 mL), water(7 mL) and glacial acetic acid (21 mL) was stirred overnight at roomtemperature. The reaction was diluted with ethyl acetate (200 mL) andwashed with water (100 mL), saturated aqueous NaHCO₃ mixture (100 mL),brine (100 mL), dried over magnesium sulfate, filtered, andconcentrated. The residue was purified by silica gel chromatography,eluting with a gradient of 0.5% to 5% methanol in dichloromethane, togive the title compound. MS (ESI) m/e 860.4 (M+H)⁺.

2.123.19(2S,3S,4R,5S,6S)-2-(5-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenethyl)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate

To a mixture of Example 2.123.18 (4.00 g) and bis(4-nitrophenyl)carbonate (2.83 g) in acetonitrile (80 mL) was addedN-ethyl-N-isopropylpropan-2-amine (1.22 mL) at room temperature. Afterstirring overnight, the reaction was concentrated, dissolved indichloromethane (250 mL) and washed with saturated aqueous NaHCO₃mixture (4×150 mL). The organic layer was dried over magnesium sulfate,filtered, and concentrated. The resulting foam was purified by silicagel chromatography, eluting with a gradient of 5% to 75% ethyl acetatein hexanes to give the title compound. MS (ESI) m/e 1025.5 (M+H)⁺.

2.123.203-(1-((3-(2-((((4-((R)-2-((R)-2-amino-3-methylbutanamido)propanamido)-2-(2-((2S,3R,4R,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)ethyl)benzyl)oxy)carbonyl)(2-sulfoethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid

To a cold (0° C.) mixture of Example 2.123.19 (70 mg) and Example 1.2.9(58.1 ng) in N,N-dimethylformamide (4 mL) was addedN-ethyl-N-isopropylpropan-2-amine (0.026 mL). The reaction was slowlywarmed to room temperature and stirred overnight. To the reactionmixture was added water (1 mL) and LiOH H₂O (20 mg). The mixture wasstirred at room temperature for 3 hours. The mixture was acidified withtrifluoroacetic acid, filtered and purified by reverse-phase HPLC on aGilson system (C18 column), eluting with 20-80% acetonitrile in watercontaining 0.1% trifluoroacetic acid, to give the title compound. MS(ESI) m/e 1564.4 (M−H)⁻.

2.123.21(6S)-2,6-anhydro-6-(2-{2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.13,7]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-5-({N-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L-valyl-L-alanyl}amino)phenyl}ethyl)-L-gulonicAcid

The title compound was prepared as described in Example 2.54, replacingExample 2.49.1 with Example 2.123.20. ¹H NMR (500 MHz, dimethylsulfoxide-d₆) δ ppm 12.86 (s, 1H), 9.92 (d, 1H), 8.35-8.19 (m, 2H), 8.04(d, 1H), 7.80 (d, 1H), 7.61 (d, 1H), 7.57-7.32 (m, 8H), 7.28 (s, 1H),7.22 (d, 1H), 7.08 (s, 2H), 6.95 (d, 1H), 5.12-4.91 (m, 5H), 4.39 (t,1H), 4.32-4.19 (m, 1H), 4.12 (s. 2H), 3.89 (t, 2H), 3.80 (d, 2H), 3.14(t, 1H), 3.06-2.87 (m, 4H), 2.69-2.58 (m, 4H), 2.37 (p, 1H), 2.09 (d,4H), 2.04-1.91 (m, 4H), 1.54 (d, 1H), 1.40-0.99 (m, 20H), 0.99-0.74 (m,16H). MS (ESI) m/e 1513.5 (M−H)⁻.

2.124 Synthesis of3-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]({[4-(4-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}butyl)-2-(beta-D-glucopyranuronosyloxy)benzyl]oxy}carbonyl)amino}propylbeta-D-glucopyranosiduronic Acid (Synthon ZM) 2.124.1A(9H-fluoren-9-yl)methyl but-3-yn-1-ylcarbamate

A mixture of but-3-yn-1-amine hydrochloride (9 g) andN,N-diisopropylethylamine (44.7 mL) was stirred in dichloromethane (70mL) and cooled to 0° C. A mixture of (9H-fluoren-9-yl)methylcarbonochloridate (22.06 g) in dichloromethane (35 mL) was added, andthe reaction stirred for 2 hours. The reaction was concentrated, and theresidue purified by silica gel chromatography, eluting with petroleumether in ethyl acetate (10%-25%) to give the title compound. MS (ESI)m/e 314 (M+Na)⁺.

2.124.1B(3R,4S,5S,6S)-2-(2-formyl-5-iodophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate

To a stirred solution of 2-hydroxy-4-iodobenzaldehyde (0.95 g) inacetonitrile (10 ml) was added(3R,4S,5S,6S)-2-bromo-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (2.5 g) and silver oxide (2 g). The mixture was covered withaluminum foil and was stirred at room temperature overnight. Afterfiltration through diatomaceous earth, the filtrate was washed withethyl acetate, the solution was concentrated. The reaction mixture waspurified by flash chromatography using an ISCO CombiFlash system,SF40-80 g column, eluted with 15-30% ethyl acetate/heptane (flow rate:60 ml/min), to provide the title compound. MS (ESI) m/e 586.9 (M+Na)⁺.

2.124.2 (2S,3S,4S,5R,6S)-methyl6-(5-(4-(((9H-fluoren-9-yl)methoxy)carbonylamino)but-1-ynyl)-2-formylphenoxy)-3,4,5-triacetoxy-tetrahydro-2H-pyran-2-carboxylate

Example 2.124.1B (2.7 g). Example 2.124.1A (2.091 g),bis(triphenylphosphine)palladium(II) chloride (0.336 g) and copper(I)iodide (0.091 g) were weighed into a vial and flushed with a stream ofnitrogen. Triethylamine (2.001 mL) and tetrahydrofuran (45 mL) wereadded, and the reaction stirred at room temperature. After stirring for16 hours, the reaction was diluted with ethyl acetate (200 mL) andwashed with water (100 mL) and brine (100 mL). The organic layer wasdried over magnesium sulfate, filtered, and concentrated. The residuewas purified by silica gel chromatography, eluting with petroleum etherin ethyl acetate (10%-50%), to give the title compound. MS (ESI) m/e 750(M+Na)⁺.

2.124.3 (2S,3S,4S,5R,6S)-methyl6-(5-(4-(((9H-fluoren-9-yl)methoxy)carbonylamino)butyl)-2-formylphenoxy)-3,4,5-triacetoxy-tetrahydro-2H-pyran-2-carboxylate

Example 2.124.2 (1.5 g) and tetrahydrofuran (45 mL) were added to 10%Pd-C (0.483 g) in a 100 mL pressure bottle and stirred for 16 hoursunder 1 atm H₂ at room temperature. The reaction was filtered andconcentrated to give the title compound. MS (ESI) m/e 754 (M+Na)⁺.

2.124.4 (2S,3S,4S,5R,6S)-methyl6-(5-(4-(((9H-fluoren-9-yl)methoxy)carbonylamino)butyl)-2-(hydroxymethyl)phenoxy)-3,4,5-triacetoxy-tetrahydro-2H-pyran-2-carboxylate

A mixture of Example 2.124.3 (2.0 g) in tetrahydrofuran (7.00 mL) andmethanol (7 mL) was cooled to 0° C., and NaBH₄ (0.052 g) was added inone portion. After 30 minutes, the reaction was diluted with ethylacetate (150 mL) and water (100 mL). The organic layer was separated,washed with brine (100 mL), dried over magnesium sulfate, filtered, andconcentrated. The residue was purified by silica gel chromatography,eluting with petroleum ether in ethyl acetate (10%-40%), to give thetitle compound. MS (ESI) m/e 756 (M+Na)⁺.

2.124.5 (2S,3S,4S,5R,6S)-methyl6-(5-(4-(((9H-fluoren-9-yl)methoxy)carbonylamino)butyl)-2-(((4-nitrophenoxy)carbonyloxy)methyl)phenoxy)-3,4,5-triacetoxy-tetrahydro-2H-pyran-2-carboxylate

To a mixture of Example 2.124.4 (3.0 g) and bis(4-nitrophenyl) carbonate(2.488 g) in dry acetonitrile (70 mL) at 0° C. was addedN,N-diisopropylethylamine (1.07 mL). After stirring at room temperaturefor 16 hours, the reaction was concentrated to give the residue, whichwas purified by silica gel chromatography, eluting with petroleum etherin ethyl acetate (10%-50%), to give the title compound. MS (ESI) m/e 921(M+Na)⁺.

2.124.63-(1-((3-(2-((((4-(4-aminobutyl)-2-(((2R,3S,4R,5R,6R)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)(3-(((2S,3S,4R,5R,6R)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)propyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)naphthalen-2-yl)picolinicAcid

To a cold (0° C.) mixture of Example 2.124.5 (44 mg) and Example 1.87.3(47.4 mg) in N,N-dimethylformamide (4 mL) was addedN-ethyl-N-isopropylpropan-2-amine (0.026 mL). The reaction was slowlywarmed to room temperature and stirred overnight. To the reactionmixture was added water (1 mL) and LiOH H₂O (20 mg). The mixture wasstirred at room temperature for 3 hours. The mixture was acidified withtrifluoroacetic acid, filtered and purified by reverse-phase HPLC on aGilson system (C18 column), eluting with 20-80% acetonitrile in watercontaining 0.1% trifluoroacetic acid, to give the title compound. MS(ESI) n/e 1564.4 (M−H)⁻.

2.124.73-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.13,7]dec-1-yl}oxy)ethyl]({[4-(4-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}butyl)-2-(beta-D-glucopyranuronosyloxy)benzyl]oxy}carbonyl)amino}propylbeta-D-glucopyranosiduronic Acid

The title compound was prepared as described in Example 2.5.4, replacingExample 2.5.3 with Example 2.124.6. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 13.06 (s, 2H), 8.99 (s, 1H), 8.34 (dd, 1H),8.25-8.09 (m, 3H), 8.08-8.02 (m, 1H), 7.98 (d, 1H), 7.89 (d, 1H), 7.78(d, 1H), 7.66 (q, 2H), 7.50-7.41 (m, 2H), 7.37-7.31 (m, 1H), 7.14 (t,1H), 6.94 (s, 2H), 6.90 (s, 1H), 6.82 (d, 1H), 5.14-5.02 (m, 2H), 4.97(d, 1H), 4.19 (d, 1H), 3.85 (dd, 3H), 3.37-3.23 (m, 9H), 3.14 (t, 1H),3.04-2.92 (m, 4H), 2.19 (s, 3H), 1.96 (t, 2H), 1.73 (s, 2H), 1.55-0.87(m, 21H), 0.81 (d, 6H). MS (ESI) m/e 1564.4 (M−H)⁻.

2.125 Synthesis ofN-{[(3S,5S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-(methoxymethyl)-2-oxopyrrolidin-1-yl]acetyl}-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide(Synthon SV) 2.125.1 Tert-butyl2-((3S,5S)-3-(dibenzylamino)-5-(methoxymethyl)-2-oxopyrrolidin-1-yl)acetate

To a mixture of Example 2.119.10 (1.4 g) in N,N-dimethylformamide (5 mL)was added iodomethane (0.8 mL). The reaction was cooled to 0° C., and95% sodium hydride (80 mg) was added. After five minutes the coolingbath was removed, and the reaction stirred at room temperature for 2.5hours. The reaction was quenched by the addition of water (20 mL) andethyl acetate (40 mL). The layers were separated, and the organic layerwas washed with brine. The combined aqueous layers were back-extractedwith ethyl acetate (10 mL). The combined organic layers were dried withsodium sulfate, filtered and concentrated under reduced pressure. Theresidue was purified by silica gel chromatography, eluting with 80/20heptane/ethyl acetate, to give the title compound. MS (DCI) m/e 439.2(M+H)⁺.

2.125.2 Tert-butyl2-((3S,5S)-3-amino-5-(methoxymethyl)-2-oxopyrrolidin-1-yl)acetate

To a mixture of Example 2.125.1 (726 mg) in 2,2,2-trifluoroethanol (10mL) was added palladium hydroxide on carbon (20/by wt, 150 mg). Thereaction was stirred under a hydrogen atmosphere (50 psi) at roomtemperature for two hours. The reaction was filtered and concentrated togive the title compound. MS (DCI) m/e 259.0 (M+H)⁺.

2.125.34-(((3S,5S)-1-(2-(tert-butoxy)-2-oxoethyl)-5-(methoxymethyl)-2-oxopyrrolidin-3-yl)amino)-4-oxobut-2-enoicAcid

The title compound was prepared by substituting Example 2.125.2 forExample 2.119.12 in Example 2.119.13. MS (DCI) m/e 374.0 (M+NH₃+H)⁺.

2.125.4 Tert-butyl2-((3S,5S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-(methoxymethyl)-2-oxopyrrolidin-1-yl)acetate

The title compound was prepared by substituting Example 2.125.3 forExample 2.119.13 in Example 2.119.14. MS (DCI) m/e 356.0 (M+NH₃+H)⁺.

2.125.52-((3S,5S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-(methoxymethyl)-2-oxopyrrolidin-1-yl)aceticacid

To a mixture of Example 2.125.4 (120 mg) in dichloromethane (8 mL) wasadded trifluoroacetic acid (4 mL). The reaction was stirred at roomtemperature for 90 minutes and then concentrated under reduced pressure.The residue was dissolved in acetonitrile (4 mL) and purified bypreparative reverse-phase HPLC with a Luna C18(2) AXIA column, 250×50mm, 10s particle size, using a gradient of 5-75% acetonitrile in 0.1%trifluoroacetic acid in water over 30 minutes, to give the titlecompound. MS (DCI) m/e 300.0 (M+NH₃+H)⁺.

2.125.6N-{[(3S,5S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-(methoxymethyl)-2-oxopyrrolidin-1-yl]acetyl}-L-valyl-N-{4-[({[2-({3-[(4-[6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.13,7]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl]-N5-carbamoyl-L-ornithinamide

The title compound was prepared by substituting Example 2.125.5 forExample 2.119.15 and Example 2.49.1 for Example 2.119.16 in Example2.119.17. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ 5 ppm 9.98 (s, 1H),8.19 (br d, 1H), 8.03 (d, 1H), 7.96 (d, 1H), 7.79 (d, 1H), 7.61 (m. 3H),7.55 (d, 1H), 7.45 (m, 2H), 7.37 (m, 2H), 7.32 (s, 1H), 7.27 (d, 2H),7.08 (s, 2H), 6.96 (d, 1H), 5.00 (m, 2H), 4.96 (s, 2H), 4.69 (t, 1H),4.39 (br m, 1H), 4.28 (m, 1H), 4.20 (d, 1H), 3.88 (t, 3H), 3.81 (br m.3H), 3.46 (m, 3H), 3.40 (m, 2H), 3.26 (br m, 2H), 3.25 (s, 3H), 3.01 (m,3H), 2.96 (m, 1H), 2.65 (t, 2H), 2.36 (br m, 1H), 2.10 (s, 3H), 2.00 (m,1H), 1.94 (m, 1H), 1.69 (br m, 1H), 1.59 (br m, 1H), 1.49-0.92 (m, 16H),0.88 (d, 3H), 0.83 (m, 9H). MS (ESI) m/e 1521.5 (M−H)⁻.

2.126 Synthesis of(6S)-2,6-anhydro-6-(2-{2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-5-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-L-alanyl}amino)phenyl}ethyl)-L-gulonicAcid (Synthon SY) The title compound was prepared as described inExample 2.123.21, replacing 2,5-dioxopyrrolidin-1-yl2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate with2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate. ¹H NMR (501 MHz,dimethyl sulfoxide-d₆) δ 6 ppm 12.83 (s, 1H), 9.87 (s, 1H), 8.09 (d,1H), 8.05-7.95 (m, 1H), 7.77 (d, 2H), 7.59 (d, 1H), 7.55-7.31 (m 7H),7.28 (s, 1H), 7.20 (d, 1H), 6.97 (s, 2H), 6.94 (d. 1H), 5.08-4.84 (m,5H), 4.36 (p, 1H), 3.78 (d, 2H), 3.54 (t, 1H), 3.48-3.28 (m, 9H), 3.21(s, 2H), 3.12 (t, 2H), 3.02-2.84 (m, 4H), 2.81-2.54 (m, 6H), 2.19-1.84(m, 9H), 1.63-1.39 (m, 6H), 1.35 (s, 1H), 1.29-0.86 (m, 18H), 0.80 (td,15H). MS (ESI) m/e 1568.4 (M−H)⁻. 2.127 Synthesis of2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)-]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-5-(4-{[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]amino}butyl)phenylbeta-D-glucopyranosiduronic Acid (Synthon TK) 2.127.13-(1-((3-(2-((((4-(4-aminobutyl)-2-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)(2-sulfoethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid

To a mixture of Example 1.2.9 (0.030 g), Example 2.124.5 (0.031 g) and1H-benzo[d][1,2,3]triazol-1-ol hydrate (5 mg) in N,N-dimethylformamide(0.5 mL) was added N-ethyl-N-isopropylpropan-2-amine (0.017 mL), and thereaction mixture was stirred for 3 hours. The reaction mixture wasconcentrated, dissolved in tetrahydrofuran (0.4 mL) and methanol (0.4mL) and treated with lithium hydroxide hydrate (0.020 g) as a mixture inwater (0.5 mL). After 1 hour, the reaction was quenched with2,2,2-trifluoroacetic acid (0.072 mL), diluted withN,N-dimethylformamide:water (1:1) (1 mL) and purified by preparatoryreverse-phase HPLC using a Gilson PLC 2020 system, eluting with agradient of 5% to 75% acetonitrile/water. Product-containing fractionswere combined and lyophilized to give to title compound. MS (ESI) m/e1251.7 (M+H)⁺.

2.127.22-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.13,7]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-5-(4-{[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]amino}butyl)phenylbeta-D-glucopyranosiduronic acid

To a mixture of Example 2.127.1 (0.027 g) and 2,5-dioxopyrrolidin-1-yl3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate (6.32 mg) inN,N-dimethylformamide (0.4 mL) was addedN-ethyl-N-isopropylpropan-2-amine (0.017 mL), and the reaction wasstirred for 1 hour at room temperature. The reaction was quenched with amixture of 2,2,2-trifluoroacetic acid (0.038 mL), water (1.5 mL) andN,N-dimethylformamide (0.5 mL) and purified by preparatory reverse-phaseHPLC on a Gilson 2020 system, using a gradient of 5% to 75%acetonitrile/water. The product-containing fractions were lyophilized togive the title compound. ¹H NMR (501 MHz, dimethyl sulfoxide-d₆) δ 12.84(s, 1H), 8.03 (dd, 1H), 7.91-7.85 (m, 1H), 7.78 (d, 1H), 7.61 (dd, 1H),7.52 (dd, 1H), 7.50-7.40 (m, 2H), 7.39-7.31 (m, 2H), 7.31 (s, 1H), 7.17(dd, 1H), 6.99-6.90 (m, 4H), 6.83 (d, 1H), 5.15-5.04 (m, 2H), 5.05-4.96(m, 1H), 4.95 (s, 2H), 3.91-3.83 (m, 4H), 3.81 (d, 3H), 3.58 (t, 2H),3.42 (td, 3H), 3.33-3.24 (m, 5H), 3.00 (q, 4H), 2.68 (dt, 2H), 2.29 (t,2H), 2.09 (d, 3H), 1.49 (d, 3H), 1.34 (td, 5H), 1.21 (dd, 5H), 1.15-1.07(m, 2H), 1.07 (s, 4H), 0.95 (q, 1H), 0.82 (d, 6H). MS (ESI) m/e 1402.1(M+H)⁺.

2.128 Synthesis of2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-5-[4-({(2S)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-[4-(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yloxy)phenyl]propanoyl}amino)butyl]phenylbeta-D-glucopyranosiduronic Acid (Synthon TR)

A mixture of Example 2.120.5 (0.035 g),0-(7-azabenotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (0.015 g) and N-ethyl-N-isopropylpropan-2-amine(0.015 mL) was stirred in N,N-dimethylformamide (0.4 mL) for 5 minutes.The mixture was added to a mixture of Example 2.127.1 (0.030 g) andN-ethyl-N-isopropylpropan-2-amine (0.015 mL) in N,N-dimethylformamide(0.4 mL) and stirred at room temperature for 3 hours. The reaction wasdiluted with a mixture of water (1.5 mL), N,N-dimethylformamide (0.5 mL)and 2,2,2-trifluoroacetic acid (0.034 mL) and purified by preparatoryreverse-phase HPLC on a Gilson 2020 system, using a gradient of 5% to85% acetonitrile/water. The product-containing fractions werelyophilized to give the title compound. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ 6 12.83 (s, 1H), 8.04-7.93 (m, 2H), 7.76 (d, 1H), 7.58(dd, 1H), 7.53-7.36 (m, 3H), 7.37-7.25 (m, 3H), 7.15 (d, 1H), 6.97-6.88(m, 4H), 6.87 (d, 2H), 6.85-6.77 (m, 1H), 6.76-6.69 (m, 2H), 5.13-4.96(m, 3H), 4.92 (s, 2H), 3.95 (dd, 2H), 3.84 (d, 2H), 3.78 (s, 8H),3.69-3.60 (m, 2H), 3.47 (d, 38H), 3.48-3.35 (m, 6H), 3.20 (s, 8f), 3.10(dd, 2H), 2.98 (t, 2H), 2.69-2.60 (m, 2H), 2.50 (d, 1H), 2.06 (s, 3H),1.49 (t, 2H), 1.35 (s, 4H), 1.21 (d, 4H), 1.05 (s, 6H), 0.79 (d, 6H). MS(ESI) m/e 1991.6 (M−H)⁻.

2.129 Synthesis of(6S)-2,6-anhydro-6-(2-{2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-5-[(N-{(2S)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-[4-(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yloxy)phenyl]propanoyl}-L-valyl-L-alanyl)amino]pheny}ethyl)-L-gulonicAcid (Synthon TY)

A mixture of Example 2.120.5 (0.033 g),O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (0.014 g) and N-ethyl-N-isopropylpropan-2-amine(0.015 mL) was stirred in N,N-dimethylformamide (0.4 mL) for 5 minutes.This mixture was added to a mixture of Example 2.123.20 (0.032 g) andN-ethyl-N-isopropylpropan-2-amine (0.015 mL) in N,N-dimethylformamide(0.4 mL) and stirred at room temperature for 3 hours. The reaction wasdiluted with a mixture of water (1.5 mL), N,N-dimethylformamide (0.5 mL)and 2,2,2-trifluoroacetic Acid (0.033 mL) and purified by preparatoryreverse-phase HPLC on a Gilson 2020 system, using a gradient of 5% to85% acetonitrile/water. The product-containing fractions werelyophilized to give the title compound. ¹H NMR (501 MHz, dimethylsulfoxide-d₆) δ 9.90 (d, 1H), 8.25 (d, 1H), 8.12 (m, 1H), 8.01 (m, 1H),1.78 (m, 1H), 7.59 (d, 1H), 7.53-7.40 (m, 4H), 7.43-7.30 (m, 4H), 7.27(s, 1H), 7.18 (d, 2H), 7.06 (s, 1H), 7.00 (d, 2H), 6.97-6.91 (m, 2H),6.87 (s, 2H), 6.76 (d, 2H), 5.02-4.92 (m, 4H), 4.77 (dd, 1H), 4.20 (t,1H), 3.98 (dd, 2H), 3.86 (t, 2H), 3.78 (d, 2H), 3.70-3.65 (m, 2H), 3.54(s, 2H), 3.55-3.45 (m, 38H), 3.45-3.37 (m, 2H), 3.35-3.25 (m, 2H), 3.21(s, 4H), 3.17-3.06 (m, 2H), 2.99 (t, 2H), 2.73 (s, 2H), 2.61 (s, 4H),2.07 (d, 4H), 2.01 (s, 2H), 1.94 (s, 2H), 1.54 (s, 2H), 1.27 (d, 4H),1.22 (s, 2H), 1.11 (s, 6H), 1.08-0.99 (m, 2H), 0.90-0.79 (m, 6H), 0.76(d, 6H). MS (ESI) m/e 705.6 (M−3H)³⁻.

2.130 Synthesis of6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-(2-((((2-(2-((2S,3R,4R,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)ethyl)-4-((S)-2-((S)-2-(2-((3S,5S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-5-((2-sulfoethoxy)methyl)pyrrolidin-1-yl)acetamido)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)(2-sulfoethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinicAcid (Synthon TX)

The title compound was prepared by substituting Example 2.123.20 forExample 2.119.16 in Example 2.119.17. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 9.85 (s, 1H), 8.17 (br d, 1H), 8.01 (d, 2H), 7.77(d, 1H), 7.59 (d, 1H), 7.53 (d, 1H), 7.43 (m, 4H), 7.34 (m, 3H), 7.19(d, 1H), 7.06 (s, 2H), 6.96 (d, 1H), 4.99 (m, 2H), 4.95 (s, 2H), 4.63(t, 1H), 4.36 (t, 1H), 4.19 (br m, 1H), 4.16 (d, 1H), 3.98 (d, 1H), 3.87(br t, 2H), 3.81 (br d, 2H), 3.73 (brm, 1H), 3.63 (t, 2H), 3.53 (m, 2H),3.44 (m, 4H), 3.31 (t, 2H), 3.21 (br m, 2H), 3.17 (m, 2H), 3.00 (m, 2H),2.92 (br m, 1H), 2.75 (m, 3H), 2.65 (br m, 3H), 2.35 (br m, 1H), 2.07(s, 3H), 1.98 (br m, 2H), 1.85 (m, 1H), 1.55 (br m, 1H), 1.34 (br m,1H), 1.26 (br m, 6H), 1.09 (br m, 7H), 0.93 (br m, 1H), 0.87, 0.83, 0.79(all d, total 12H). MS (ESI) m/e 1733.4 (M−H)⁻.

2.131 Synthesis of6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-(2-((((2-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-4-(4-(2-((3S,5S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-5-((2-sulfoethoxy)methyl)pyrrolidin-1-yl)acetamido)butyl)benzyl)oxy)carbonyl)(2-sulfoethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinicAcid (Synthon TZ)

The title compound was prepared by substituting Example 2.127.1 forExample 2.119.16 in Example 2.119.17. ¹H NMR (500 MHz, dimethylsulfoxide-d₄) δ ppm 8.02 (d, 1H), 7.82 (br t, 1H), 7.77 (d, 1H), 7.60(d, 1H), 7.53 (br d, 1H), 7.45 (ddd, 1H), 7.42 (d, 1H), 7.36 (d, 1H),7.35 (s, 1H), 7.33 (m, 1H), 7.15 (d, 1H), 7.05 (s, 2H), 6.97 (d, 1H),6.94 (s, 1H), 6.83 (d, 1H), 5.07 (br m, 2H), 5.00 (d, 1H), 4.95 (s, 2H),4.69 (t, 1H), 4.04 (d, 2H), 3.87 (m, 3H), 3.82 (m, 3H), 3.73 (br m, 1H),3.61 (m, 2H), 3.47 (br m, 3H), 3.40 (m, 4H), 3.29 (m, 4H), 3.06 (br m,2H), 3.00 (t, 2H), 2.73 (br m, 2H) 2.69 (br m, 2H), 2.52 (br t, 2H),2.35 (br m, 1H), 2.08 (s, 3H), 1.81 (m, 1H), 1.53 (br m, 2H), 1.40 (m,2H), 1.35 (br m, 2H), 1.29-0.88 (br m, 10H), 0.82, 0.80 (both s, total6H). MS (ESI-) m/e 1607.5 (M−H)⁻.

2.132 Synthesis of2-[([2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl)oxy)methyl]-5-(4-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}butyl)phenylbeta-D-glucopyranosiduronic acid (Synthon UA)

To a mixture of Example 2.127.1 (0.032 g) in N,N-dimethylformamide (0.4mL) was added N-ethyl-N-isopropylpropan-2-amine (0.025 mL), and themixture cooled to 0° C. 2,5-Dioxopyrrolidin-1-yl2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (8.86 mg) was added inone portion and stirred at 0° C. for 45 minutes. The reaction wasdiluted with a mixture of water (1.5 mL), N,N-dimethylformamide (0.5 mL)and 2,2,2-trifluoroacetic acid (0.036 mL) and was purified bypreparatory reverse-phase HPLC on a Gilson 2020 system, using a gradientof 5% to 75% acetonitrile/water. The product-containing fractions werelyophilized to give the title compound. ¹H NMR (501 MHz, dimethylsulfoxide-d₆) δ 12.86 (s, 1H), 8.06 (s, 1H), 8.02 (dd, 1H), 7.77 (d,1H), 7.60 (dd, 1H), 7.51 (dd, 1H), 7.49-7.39 (m, 2H), 7.38-7.28 (m, 3H),7.17 (dd, 1H), 7.06 (d, 2H), 6.98-6.89 (m, 2H), 6.83 (d, 1H), 5.13-5.03(m, 2H), 5.04-4.96 (m, 1H), 4.94 (s, 2H), 3.97 (s, 2H), 3.90-3.77 (m,6H), 3.50 (s, 1H), 3.50-3.41 (m, 2H), 3.41 (dt, 3H), 3.28 (dt, 4H),3.06-2.96 (m, 4H), 2.66 (dt. 2H), 2.51 (s, 2H), 2.08 (d, 3H), 1.52 (s,2H), 1.42-1.32 (m, 4H), 1.23 (d, 4H), 1.11 (q, 2H), 1.06 (s, 4H), 0.81(d, 6H). MS (ESI) m/e 1388.0 (M+H)⁺.

2.133 Synthesis of2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-5-(4-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}butyl)phenylbeta-D-glucopyranosiduronic Acid (Synthon UZ) 2.133.13-(1-((3-(2-((((4-(4-aminobutyl)-2-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)(2-sulfoethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)naphthalen-2-yl)picolinicAcid

To a mixture of Example 2.124.5 (0.060 g), Example 1.43.7 (0.056 g) and1H-benzo[d][1,2,3]triazol-1-ol (8 mg) in dimethyl sulfoxide (0.5 mL) wasadded N-ethyl-N-isopropylpropan-2-amine (0.056 mL), and the reaction wasstirred at room temperature for 3 hours. The reaction was treated with amixture of lithium hydroxide hydrate (0.026 g) in water (1 mL) andstirred for 30 minutes. Methanol (0.5 mL) was added to the reaction andstirring was continued for 30 minutes. Diethylamine (0.033 mL) was addedto the reaction and stirring was continued overnight. The reaction wasquenched with 2,2,2-trifluoroacetic acid (0.120 mL) and purified bypreparatory reverse-phase HPLC on a Gilson 2020 system, using a gradientof 5% to 75% acetonitrile/water. The product-containing fractions werelyophilized to give the title compound. MS (ESI) m/e 1247.7 (M+H)⁺.

2.133.22-[({[2-((3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.13,7]dec-1-yloxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-5-(4-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}butyl)phenylbeta-D-glucopyranosiduronic acid

To a mixture of Example 2.133.1 (0.030 g) in N,N-dimethylformamide(0.400 mL) was added N-ethyl-N-isopropylpropan-2-amine (0.023 mL) andthe mixture was cooled to 0° C. 2,5-Dioxopyrrolidin-1-yl2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (8.34 mg) was added inone portion and be mixture was stirred at 0° C. for 30 minutes. Thereaction was diluted with a mixture of water (1.5 mL),N,N-dimethylformamide (0.5 mL) and 2,2,2-trifluoroacetic acid (0.034 mL)and was purified by preparatory reverse-phase HPLC on a Gilson 2020system, using a gradient of 5% to 75% acetonitrile/water. Theproduct-containing fractions were lyophilized to give the titlecompound. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ 13.08 (s, 1H), 9.01(s, 1H), 8.39-8.31 (m, 1H), 8.25-8.11 (m, 3H), 8.06 (d, 2H), 7.99 (d,1H), 7.94 (d, 1H), 7.79 (d, 1H), 7.68 (t, 1H), 7.51-7.42 (m, 1H), 7.46(s, 1H), 7.35 (t, 1H), 7.22-7.13 (m, 1H), 7.06 (d, 2H), 6.93 (d, 1H),6.83 (d, 1H), 5.15-5.00 (m, 2H), 4.99 (d, 1H), 3.97 (s, 2H), 3.86 (d,3H), 3.42 (d, 4H), 3.29 (d, 5H), 3.03 (p, 2H), 2.72-2.62 (m, 2H), 2.51(d, 3H), 2.21 (s, 3H), 1.51 (q, 2H), 1.37 (q, 4H), 1.24 (d, 4H), 1.10(s, 5H), 0.83 (d, 6H), 0.61 (s, 2H). MS (ESI) m/e 1383.0 (M+H)⁺.

2.134 Synthesis of2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-5-[4-({(2S)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-[4-(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yloxy)phenyl]propanoyl}amino)butyl]phenylbeta-D-glucopyranosiduronic Acid (Synthon UK)

A mixture of Example 2.120.5 (0.028 g),O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (0.013 g) and N-ethyl-N-isopropylpropan-2-amine(0.015 mL) were stirred in N,N-dimethylformamide (0.4 mL) for 5 minutes.The mixture was added to a mixture of Example 2.133.1 (0.030 g) andN-ethyl-N-isopropylpropan-2-amine (0.015 mL) in N,N-dimethylformamide(0.4 mL) and was stirred at room temperature for 1 hour. The reactionwas diluted with a mixture of water (1.5 mL). N,N-dimethylformamide (0.5mL) and 2,2,2-trifluoroacetic acid (0.042 mL) and was purified bypreparatory reverse-phase HPLC on a Gilson 2020 system, using a gradientof 5% to 75% acetonitrile/water. The product-containing fractions werelyophilized to give the title compound. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ 9.01 (s, 1H), 8.35 (dd, 1H), 8.27-8.13 (m, 3H), 8.06 (d,1H), 8.00 (d, 1H), 7.94 (d, 1H), 7.79 (d, 1H), 7.73-7.64 (m, 1H),7.53-7.43 (m, 2H), 7.42-7.32 (m, 1H), 7.17 (d, 1H), 7.06 (s, 1H),7.04-6.91 (m, 3H), 6.89 (d, 2H), 6.83 (d, 1H), 6.74 (d, 1H), 5.16-4.93(m, 4H), 4.63 (dd, 2H), 3.96 (t, 2H), 3.86 (d, 4H), 3.66 (s, 4H),3.55-3.46 (m, 36H), 3.45-3.35 (m, 8H), 3.35-3.24 (m, 6H), 3.21 (s, 2H),3.11 (s, 2H), 2.99 (d, 2H), 2.83-2.59 (m, 3H), 2.52 (d, 2H), 2.21 (s,3H), 1.57-0.86 (m, 14H), 0.83 (d, 4H). MS (ESI) m/1986.6 (M−H)⁻.

2.135 Synthesis ofN-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-3-(4-carboxybutyl)phenyl}-L-alaninamide(Synthon UU) 2.135.1 Methyl4-((tert-butoxycarbonyl)amino)-2-iodobenzoate

3-Iodo-4-(methoxycarbonyl)benzoic acid (9 g) was dissolved intert-butanol (100 mL), and diphenyl phosphorazidate (7.6 mL) andtriethylamine (4.9 mL) were added. The mixture was heated to 83° C.(internal temperature) overnight. The mixture was concentrated todryness and purified by flash chromatography, eluting with a gradient of0% to 20% ethyl acetate in heptane to give the title compound. MS (ESI)m/e 377.9 (M+H)⁺.

2.135.2 Methyl 4-amino-2-iodobenzoate

Example 2.135.1 (3 g) was stirred in dichloromethane (30 mL) andtrifluoroacetic acid (10 mL) at room temperature for 1.5 hours. Thereaction was concentrated to dryness and partitioned between water(adjusted to pH 1 with hydrochloric acid) and diethyl ether. The layerswere separated, and the aqueous layer was washed with aqueous sodiumbicarbonate mixture, dried over sodium sulfate, filtered andconcentrated to dryness. The resulting solid was triturated with tolueneto give the title compound. MS (ESI) m/e 278.0 (M+H)⁺.

2.135.3 Methyl4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)-2-iodobenzoate

A flask was charged with Example 2.135.2 (337 mg) and Example 2.123.14(500 mg). Ethyl acetate (18 mL) was added followed by pyridine (0.296mL). The resulting suspension was chilled in an ice bath, and2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide (50%mixture in ethyl acetate. 1.4 mL) was added dropwise. Stirring wascontinued at 0° C. for 45 minutes, and the reaction was placed in a −20°C. freezer overnight. The reaction was allowed to warm to roomtemperature and was quenched with water. The layers were separated, andthe aqueous layer was extracted twice more with ethyl acetate. Thecombined extracts were dried with anhydrous sodium sulfate, filtered andconcentrated. The residue was dissolved in dichloromethane and dilutedwith diethyl ether to precipitate the title compound, which wascollected by filtration. MS (ESI) m/e 669.7 (M+H)⁺.

2.135.4 (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-(hydroxymethyl)-3-iodophenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate

Example 2.54.3 (1 g) was dissolved in tetrahydrofuran (15 mL), and themixture was chilled to −15° C. in an ice-acetone bath. Lithium aluminumhydride (1N in tetrahydrofuran, 3 mL) was then added dropwise, keepingthe temperature below −10° C. The reaction was stirred for 1 hour andcarefully quenched with 10% citric acid (25 mL). The layers wereseparated, and the aqueous layer was extracted thrice with ethylacetate. The combined organic layers were washed with water and brine,dried over anhydrous sodium sulfate, filtered and concentrated. Theresidue was adsorbed onto silica gel and purified by flashchromatography, eluting with a gradient of 5% to 6% methanol indichloromethane, to give the title compound. MS (ESI) m/e 664.1 (M+H)⁺.

2.135.5 methyl5-(5-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)-2-(hydroxymethyl)phenyl)pent-4-ynoate

To a stirred mixture of methyl pent-4-ynoate (50 mg), Example 2.135.4(180 mg) and N,N-diisopropylethylamine (0.15 mL) inN,N-dimethylformamide (2 mL) was addedbis(triphenylphosphine)palladium(II) dichloride (20 mg) and copperiodide (5 mg). The mixture was purged with nitrogen three times andstirred at room temperature overnight. The reaction was diluted withethyl acetate and washed with water and brine. The aqueous layers wereback extracted with ethyl acetate. The combined organic layers weredried over sodium sulfate, filtered and concentrated. The residue waspurified by reverse-phase HPLC on a Gilson system, eluting with 20-90%acetonitrile in water containing 0.1% v/v trifluoroacetic acid. Thedesired fractions were combined and freeze-dried to provide the titlecompound. MS (ESI) m/e 608.0 (M−H₂O)⁺.

2.135.6 methyl5-(5-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)-2-(hydroxymethyl)phenyl)pentanoate

A mixture of Example 2.135.5 (0.084 g) and 10% Pd/C (0.02 g) intetrahydrofuran (5 mL) was stirred at 20° C. under an atmosphere of 50psi H₂ for 1 hour. The reaction mixture was filtered throughdiatomaceous earth, and the solvent was evaporated under reducedpressure to provide the title compound. MS (ESI) m/e 612.0 (M−H₂O)⁺.

2.135.7 methyl5-(5-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)pentanoate

Example 2.135.7 was prepared by substituting Example 2.135.7 for Example2.55.6 in Example 2.55.7. MS (ESI) m/e 795.4 (M+H)⁺.

2.135.83-(1-((3-(2-((((4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)-2-(4-carboxybutyl)benzyl)oxy)carbonyl)(2-sulfoethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid

Example 2.135.8 was prepared by substituting 2.135.7 for(9H-fluoren-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-1-oxobutan-2-yl)carbamatein Example 2.49.1. MS (ESI) m/e 1271.4 (M−H)⁻.

2.135.9N-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.13,7]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-3-(4-carboxybutyl)phenyl}-L-alaninamide

Example 2.135.9 was prepared by substituting 2.135.8 for Example 2.49.1in Example 2.54. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm 9.88 (d,1H), 8.3-8.2 (m, 2H), 8.01 (dd, 1H), 7.77 (d, 1H), 7.59 (dd, 1H), 7.52(dd, 1H), 7.47-7.29 (m, 8H), 7.23-7.18 (m, 1H), 7.05 (s, 2H), 6.95 (d,1H), 5.00 ((d, 2H), 4.94 (s, 2H), 4.37 (p, 1H), 3.51-3.28 (m, 5H),3.26-3.14 (m, 2H), 2.99 (t, 2H), 2.65 (t, 2H), 2.57 (s, 2H), 2.26-2.17(m, 3H), 2.07 (d, 3H), 1.94 (dd, 1H), 1.61-0.69 (m, 35H). MS (ESI) m/e1408.5 (M−H)⁺.

2.136 Synthesis of2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-5-(3-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}propyl)phenylbeta-D-glucopyranosiduronic Acid (Synthon UV) 2.136.1(3R,4S,5S,6S)-2-(5-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)prop-1-yn-1-yl)-2-formylphenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-34,5-triyltriacetate

Example 2.136.1 was prepared by substituting (9H-fluoren-9-yl)methylprop-2-yn-1-ylcarbamate for 2.124.1A in Example 2.124.2. MS (ESI) m/e714.1 (M+H)⁺.

2.136.2(2S,3R,4S,5S,6S)-2-(5-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propyl)-2-formylphenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate

Example 2.136.2 was prepared by substituting 2.136.1 for 2.124.2 inExample 2.124.3. MS (ESI) m/e 718.5 (M+H)⁺.

2.136.3(2S,3R,4S,5S,6S)-2-(5-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propyl)-2-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate

Example 2.136.3 was prepared by substituting 2.136.2 for 2.124.3 inExample 2.124.4. MS (ESI) m/e 742.2 (M+Na)⁺. 2.136.4(2S,3R,4S,5S,6S)-2-(5-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propyl)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate Example 2.136.4 was prepared by substituting 2.136.3 for2.124.4 in Example 2.124.5. MS (ESI) m/e 885.2 (M+Na)⁺.

2.136.53-(1-((3-(2-((((4-(3-aminopropyl)-2-(((3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)(2-sulfoethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicacid

Example 2.136.5 was prepared by substituting Example 2.136.4 for(9H-fluoren-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-1-oxobutan-2-yl)carbamatein Example 2.49.1. MS (ESI) m/e 1237.7 (M+H)⁺.

2.136.62-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.13,7]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-5-(3-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}propyl)phenylbeta-D-glucopyranosiduronic acid

Example 2.136.6 was prepared by substituting Example 2.136.5 for Example2.49.1 in Example 2.54. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm8.14 (d, 1H), 8.01 (d, 1H), 7.59 (d. 1H), 7.53-7.39 (m, 4H), 7.38-7.28(m, 3H), 7.22-7.15 (m, 2H), 7.13-6.91 (m, 5H), 6.84 (d, 1H), 5.17-4.91(m, 5H), 3.35-3.2 (m, 4H), 3.10-2.90 (m, 4H), 2.75-2.65 (m, 2H), 2.08(s, 3H), 1.65 (s. 2H), 1.39-0.71 (m, 21H). MS (ESI) m/e 1372.3 (M−H)⁻.

2.137 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[({[2-{[(2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl]oxy}-4-(4-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}butyl)benzyl]oxy}carbonyl)(3-{[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino}-3-oxopropyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (Synthon UZ) 2.137.13-(1-((3-(2-((((4-(4-aminobutyl)-2-(((2R,3S,4R,5R,6R)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)(3-((1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)amino)-3-oxopropyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicacid

The tide compound was prepared as described in Example 2.124.6,replacing Example 1.87.3 with Example 1.84. MS (ESI) m/e 1319.4 (M−H)⁻.

2.137.26-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-{1-[(3-{2-[({[2-[1(2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl]oxy}-4-(4-[[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino]butyl)benzyl]oxy)carbonyl)(3-([1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino}-3-oxopropyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.13,7]dec-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl)pyridine-2-carboxylicAcid

The title compound was prepared as described in Example 2.54, replacingExample 2.49.1 with Example 2.137.1. ¹H NMR (501 MHz, dimethylsulfoxide-d₆) δ ppm 12.83 (s, 2H), 8.12 (s, 0H), 8.06 (s, 1H), 8.03-7.99(m, 1H), 7.77 (d, 1H), 7.72 (s, 0H), 7.60 (d, 1H), 7.52-7.39 (m, 3H),7.34 (td, 2H), 7.26 (s, 1H), 7.21-7.11 (m, 2H), 7.05 (s, 2H), 6.93 (d,2H), 6.83 (d, 1H), 5.09 (d, 2H), 5.00 (d, 1H), 4.94 (s, 2H), 4.12 (t,1H), 3.97 (s, 2H), 3.87 (q, 4H), 3.79 (d, 2H), 3.29 (q, 2H), 3.12-2.93(m, 5H), 2.47-2.23 (m, 1H), 2.07 (d, 3H), 1.50 (d, 3H), 1.36 (d, 5H),1.31-0.85 (m, 9H), 0.81 (d, 7H). MS (ESI) m/e 1568.4 (M−H)⁻.

2.138 Synthesis of6-(8-(benzo[d]thiazol-2-ylcarbamoyl)naphthalen-2-yl)-3-(1-((3-(2-((((2-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-4-(4-(2-((3S,5S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-5-((2-sulfoethoxy)methyl)pyrrolidin-1-yl)acetamido)butyl)benzyl)oxy)carbonyl)(2-sulfoethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinicacid (Synthon VB)

The title compound was prepared by substituting Example 2.133.1 forExample 2.119.16 in Example 2.119.17. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 8.99 (s, 1H), 8.34 (dd, 1H), 8.19 (d, 1H), 8.17 (d,1H), 8.13 (d, 1H), 8.04 (d, 1H), 7.97 (d, 1H), 7.93 (d, 1H), 7.80 (br t,1H), 7.77 (d, 1H), 7.67 (dd, 1H), 7.45 (s, 1H), 7.45 (dd, 1H), 7.34 (dd,1H), 7.14 (d, 1H), 7.03 (s, 2H), 6.93 (s, 1H), 6.82 (br d, 1H), 5.06 (brm, 2H), 4.98 (d, 1H), 4.67 (t, 1H), 4.02 (d, 2H), 3.85 (m, 3H), 3.71 (brm, 1H), 3.59 (t, 2H), 3.45 (br m, 3H), 3.41 (m, 4H), 3.27 (m, 4H), 3.03(m, 2H), 2.70 (m, 2H) 2.65 (br m, 2H), 2.50 (br t, 2H), 2.31 (br m, 1H),2.19 (s, 3H), 1.80 (m, 1H), 1.52 (br m, 2H), 1.38 (m, 2H), 1.35 (br m,2H), 1.29-0.88 (br m, 10H), 0.82 (s, 3H), 0.80 (s, 3H). MS (ESI) m/e1602.4 (M−H)⁻.

2.139 Synthesis of2-[({[2-((3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl][3-hydroxy-2-(hydroxymethyl)propyl]carbamoyloxy)methyl]-5-(3-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}propyl)phenylbeta-D-glucopyranosiduronic Acid (Synthon VC) 2.139.13-(1-((3-(2-((((4-(3-aminopropyl)-2-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)(3-hydroxy-2-(hydroxymethyl)propyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicacid

Example 2.139.1 was prepared by substituting Example 2.136.4 for(9H-fluoren-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-1-oxobutan-2-yl)carbamateand substituting Example 1.79.3 for Example 1.2.9 in Example 2.49.1. MS(ESI) m/e 1217.7 (M+H)⁺.

2.139.22-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.13,7]dec-1-yl}oxy)ethyl][3-hydroxy-2-(hydroxymethyl)propyl]carbamoyl}oxy)methyl]-5-(3-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}propyl)phenylbeta-D-glucopyranosiduronic acid

Example 2.139.1 was prepared by substituting Example 2.139.1 for Example2.49.1 in Example 2.54. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm12.84 (s, 2H), 8.11 (t, 1H), 8.00 (dd, 1H), 7.76 (d, 1H), 7.62-7.56 (m,1H), 7.50-7.37 (m, 3H), 7.37-7.29 (m, 2H), 7.25 (s, 1H), 7.16 (d, 1H),7.04 (s, 2H), 6.96-6.88 (m, 2H), 6.82 (d, 1H), 5.06 (s, 2H), 4.98 (d,1H), 4.92 (s, 2H), 3.97 (s, 2H), 3.44-3.18 (m, 1H), 3.07-2.90 (m, 4H),2.05 (s, 3H), 1.80 (s, 1H), 1.64 (p, 2H), 1.38-0.67 (m, 19H). (m, 21H).MS (ESI) m/e 1352.5 (M−H)⁻.

2.140 Synthesis ofN-({(3S,5S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-5-[(2-sulfoethoxy)methyl]pyrrolidin-1-yl}acetyl)-L-valyl-N-(4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-3-(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50-heptadecaoxatripentacont-52-yn-53-yl)phenyl)-L-alaninamide(Synthon VS) 2.140.1 2-iodo-4-nitrobenzoic Acid

2-Amino-4-nitrobenzoic acid (50 g) was added to a mixture ofconcentrated H₂SO4 (75 mL) and water (750 mL) at 0° C., and the mixturewas stirred for 1 hour. To the mixture was added a mixture of sodiumnitrite (24.62 g) in water (300 mL) dropwise at 0° C. The resultingmixture was stirred at 0° C. for 3 hours. A mixture of sodium iodide(65.8 g) in water (300 mL) was added to above mixture slowly. After thecompletion of the addition, the resulting mixture was stirred at 0° C.for 2 hours, then at room temperature for 16 hours and at 60° C. for 2hours. The resulting mixture was cooled to room temperature and dilutedwith ice-water (300 mL). The solid was collected by filtration, washedby water (100 mL×5), and dried in air for 16 hours to give the titlecompound. MS (LC-MS) me 291.9 (M−H)⁻.

2.140.2 Methyl 2-iodo-4-nitrobenzoate

A mixture of Example 2.140.1 (130 g) in a mixture of methanol (1000 mL)and sulfuric acid (23.65 mL) was stirred at 85° C. for 16 hours andconcentrated to dryness. The residue was triturated with methanol (100mL) and the suspension was stirred for 10 minutes. The solid wascollected by filtration, washed with water (200 mL×3) and methanol (20mL), and air-dried for 16 hours to give the title compound. MS (LC-MS)m/e 308.0 (M+H)⁺.

2.140.3 Methyl 4-amino-2-iodobenzoate

To a mixture of ammonium chloride (122 g) and iron (38.2 g) in ethanol(1000 mL) and water (100 mL) was added Example 2.140.2 (70 g,) at roomtemperature. The mixture was stirred at 80° C. for 4 hours and filteredto remove insoluble material. The filtrate was concentrated underreduced pressure. The residue was dissolved in ethyl acetate (1000 mL)and washed with water (500 mL). The aqueous phase was extracted withethyl acetate (1000 mL×2). The combined organic phase was washed withbrine, dried over MgSO₄, filtered and concentrated to give the titlecompound. MS (LC-MS) m/e 278.0 (M+H)⁺.

2.140.4 (4-amino-2-iodophenyl)methanol

To a mixture of Example 2.140.3 (40 g) in tetrahydrofuran (800 mL) wasadded 1M diisobutylaluminum hydride (505 mL) dropwise at −50° C. Themixture was stirred at −50° C. for 3 hours and cooled to −20° C.Ice-water (180 mL) was added dropwise (keeping temperature below 0° C.)to the mixture. After the addition of ice-water, the mixture was stirredfor 10 minutes and filtered. The filtrate was concentrated, and theresidue was dissolved in ethyl acetate (800 mL) and water (200 mL). Theaqueous phase was extracted with ethyl acetate (300 mL×2). The combinedorganic phases were washed with brine, dried over MgSO₄, filtered andconcentrated to give the title compound. MS (LC-MS) me 250.0 (M+H)⁺.

2.140.5 4-(((tert-butyldimethylsilyl)oxy)methyl)-3-iodoaniline

To a mixture of Example 2.140.4 (40 g) and imidazole (21.87 g) indichloromethane (600 mL) and tetrahydrofuran (150 mL) was addedtert-butyldimethylchlorosilane (29.0 g). The mixture was stirred at roomtemperature for 16 hours and filtered to remove the solid. To thefiltrate was added ice-water (50 mL). The mixture was stirred for 10minutes and water (100 mL) was added. The mixture was extracted withdichloromethane (500 mL×2). The combined organic phases were washed withbrine, dried over MgSO₄, filtered and concentrated. The residue waspurified by silica gel chromatography, eluting with 15/1 to 10/1petroleum ether/ethyl acetate, to give the title compound. MS (LC-MS) me364.0 (M+H)⁺.

2.140.6 (S)-tert-butyl(1-((4-(((tert-butyldimethylsilyl)oxy)methyl)-3-iodophenyl)amino)-1-oxopropan-2-yl)carbamate

To a mixed mixture of (S)-2-((tert-butoxycarbonyl)amino)propanoic acid(15.62 g) and Example 2.140.5 (30 g) in dichloromethane (600 mL) at 0°C. was added POCl₃ (15.39 mL) dropwise. The mixture was stirred at 0° C.for 2 hours. Ice-water (60 mL) was carefully added to the mixturedropwise (keeping temperature below 5° C.). The mixture was stirred for30 minutes and concentrated to remove dichloromethane. The residue wassuspended in ethyl acetate (500 mL) and water (100 mL). The suspensionwas filtered. The organic phase was washed by water (200 mL×2) andbrine, dried over MgSO₄, filtered and concentrated to give the titlecompound. MS (LC-MS) m/e 533.0 (M−H)⁺.

2.140.7 (S)-tert-butyl(1-((4-(hydroxymethyl)-3-iodophenyl)amino)-1-oxopropan-2-yl)carbamate

To a mixture of Example 2.140.6 (60 g) in tetrahydrofuran (600 mL) wasadded tetrabutyl ammonium fluoride (28.2 g) in tetrahydrofuran (120 mL)at 0° C. The mixture was stirred at room temperature for 16 hours andfiltered. To the filtrate was added water (100 mL). The mixture wasstirred for 10 minutes and then concentrated. The residue was dilutedwith ethyl acetate (800 mL) and water (300 mL). The aqueous phase wasextracted with ethyl acetate (200 mL×3). The combined organic phaseswere washed with brine, dried over MgSO₄, filtered and concentrated. Theresidue was purified by silica gel chromatography, eluting with 3/1to/petroleum ether/ethyl acetate, to give the title compound. MS (LC-MS)m/e 443.0 (M+Na)⁺.

2.140.8 (S)-2-amino-N-(4-(hydroxymethyl)-3-iodophenyl)propanamide

A mixture of Example 2.140.7 (20 g) in a mixture of dichloromethane (80mL) and trifluoroacetic acid (40 mL) was stirred at room temperature for2 hours and concentrated. The residue was dissolved in dichloromethane(80 mL) and triethylamine (16.95 mL) was added to adjust the pH to 8.The title compound was obtained as free base in dichloromethane, whichwas used in next step without further purification. MS (LC-MS) m/e 321.1(M+H)⁺.

2.140.9 Tert-butyl((S)-1-(((S)-1-((4-(hydroxymethyl)-3-iodophenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate

A mixture of (S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanoic acid(6.79 g), triethylamine (9.58 mL) and 1-hydroxybenzotriazole hydrate(5.26 g) in dichloromethane (250 mL) was stirred for 20 minutes. Theresulting mixture was added to a mixture of Example 2.140.8 (10 g) and1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide hydrochloride (6.59 g)in dichloromethane (100 mL) at 0° C., dropwise. After the completion ofaddition, the mixture was stirred at 0° C. for 2 hours. Ice-water (200mL) was added, and the resulting mixture was stirred for 20 minutes. Theorganic phase was washed with saturated aqueous sodium bicarbonatemixture (100 mL×2), water (100 mL×2) and brine (100 mL), dried overMgSO₄, filtered and concentrated. The residue was purified by silica gelchromatography, eluting with 3/1 to 1/1 petroleum ether/ethyl acetate,to give the title compound. LC-MS m/e 542.1 (M+Na)⁺.

2.140.10 tert-butyl((S)-1-(((S)-1-((4-(hydroxymethyl)-3-(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50-heptadecaoxatripentacont-52-yn-53-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate

To a mixture of Example 2.140.9 (50 mg),2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50-heptadecaoxatripentacont-52-yne(149 mg), bis(triphenylphosphine)palladium(II) dichloride (27.0 mg) andN,N-diisopropylethylamine (0.05 mL) in N,N-dimethylformamide (1 mL) wasadded copper( ) iodide (3.67 mg). The reaction was purged with a streamof nitrogen gas for 10 minutes and stirred overnight. The reaction wasdiluted with dimethyl sulfoxide purified by reverse-phase HPLC on aGilson system (C18 column), eluting with 20-70% acetonitrile in watercontaining 0.1% trifluoroacetic acid, to give the title compound. MS(LC-MS) m/e 1164.2 (M−H)⁻.

2.140.11 Tert-butyl((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)-3-(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50-heptadecaoxatripentacont-52-yn-53-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxobutan-2-yl)carbamate

To a mixture of Example 2.140.10 (80 mg) and bis(4-nitrophenyl)carbonate (31.3 mg) in N,N-dimethylformamide (0.2 mL) was addedN,N-diisopropylethylamine (0.06 mL). The mixture was stirred 3 hours andwas purified by reverse-phase HPLC on a Gilson system (C18 column),eluting with 35-75% acetonitrile in water containing 0.1%trifluoroacetic acid, to give the title compound.

2.140.126-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-(2-((((4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)propanamido)-2-(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50-heptadecaoxatripentacont-52-yn-53-yl)benzyl)oxy)carbonyl)(2-sulfoethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinicAcid

To a mixture of Example 1.2.9 (95 mg). Example 2.140.11 (148 mg) and1-hydroxybenzotriazole hydrate (68.1 mg) in N,N-dimethylformamide (2.5mL) was added N,N-diisopropylethylamine (97 μL). The mixture was stirredfor 3.5 hours and purified by reverse-phase HPLC on a Gilson system (C18column), eluting with 35-80% acetonitrile in water containing 0.1%trifluoroacetic acid, to give the title compound.

2.140.133-(1-((3-(2-((((4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)-2-(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50-heptadecaoxatripentacont-52-yn-53-yl)benzyl)oxy)carbonyl)(2-sulfoethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicacid

A cold (0° C.) mixture of Example 2.140.12 (135 mg) in dichloromethane(4 mL) was treated with trifluoroacetic acid (1 mL) for 5 hours. Themixture was concentrated and purified by reverse-phase HPLC on a Gilsonsystem (C18 column), eluting with 20-60% acetonitrile in watercontaining 0.1% trifluoroacetic acid, to give the title compound. MS(ESI) m/e 973.4 (M+2H)²+.

2.140.14N-({(3S,5S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-5-[(2-sulfoethoxy)methyl]pyrrolidin-1-yl}acetyl)-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.13,7]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-3-(2,5,8,11,14,17,20,23,26,29,32,35,38,41,4447,50-heptadecaoxatripentacont-52-yn-53-yl)phenyl}-L-alaninamide

A mixture of Example 2.119.15 (20.88 mg) andO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (21.1 mg) in N,N-dimethylformamide (0.4 mL) wastreated with N,N-diisopropylethylamine (16.2 μL) for 7 minutes, and amixture of Example 2.140.13 (60 mg) and N,N-diisopropylethylamine (32.3μL) in N,N-dimethylformamide (0.6 mL) was slowly added. The reactionmixture was stirred for 10 minutes and was purified by reverse-phaseHPLC on a Gilson system (C18 column), eluting with 20-70% acetonitrilein water containing 0.1% trifluoroacetic acid, to give the titlecompound. 1H NMR (500 MHz, dimethyl sulfoxide-d6) δ 10.01 (d, 1H), 8.22(d, 1H), 8.02 (t, 2H), 7.90-7.75 (m, 2H), 7.66-7.50 (m, 3H), 7.50-7.39(m, 3H), 7.35 (q, 3H), 7.05 (s, 2H), 7.00 (d, 1H), 5.08 (d, 2H), 4.97(s, 2H), 4.65 (t, 1H), 4.47-4.31 (m, 4H), 4.23-4.14 (m, 2H), 3.90-3.69(m, 5H), 3.68-3.58 (m, 4H), 3.57-3.53 (m, 2H), 3.52-3.43 (m, 57H),3.42-3.33 (m, 4H), 3.22 (s, 5H), 3.01 (t, 2H), 2.49 (p, 3H), 2.09 (d,3H), 2.04-1.77 (m, 1H), 1.40-1.17 (m, 6H), 1.06 (dd, 6H), 0.97-0.63 (m,11H). MS (ESI) m/e 1153.3 (M+2H)²+.

2.141 Synthesis ofN-({(3S,5S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-5-[(2-sulfoethoxy)methyl]pyrrolidin-1-yl}acetyl)-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-3-(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50-heptadecaoxatripentacontan-53-yl)phenyl}-L-alaninamide(Synthon VT) 2.141.1 Tert-butyl((S)-1-(((S)-1-((3-(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50-heptadecaoxadopentacontan-52-yl)-4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate

A mixture of Example 2.140.10 (304 mg) and 10% Pd/C (90 mg, dry) intetrahydrofuran (20 mL) was shaken in a pressure bottle for 2 hoursunder 50 psi of hydrogen gas. The insoluble material was filtered off,and the filtrate was concentrated to provide the title compound. MS(ESI) m/e 1168.3 (M−H)⁻.

2.141.2 Tert-butyl((S)-1-(((S)-1-((3-(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50-heptadecaoxadopentacontan-52-yl)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate

The title compound was prepared using the procedure in Example 2.140.11,replacing Example 2.140.10 with Example 2.141.1.

2.141.36-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-(2-((((4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)propanamido)-2-(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50-heptadecaoxatripentacontan-53-yl)benzyl)oxy)carbonyl)(2-sulfoethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinicAcid

The title compound was prepared using the procedure in Example 2.140.12,replacing Example 2.140.11 with Example 2.141.2.

2.141.43-(1-((3-(2-((((4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)-2-(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50-heptadecaoxatripentacontan-53-yl)benzyl)oxy)carbonyl)(2-sulfoethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid

The title compound was prepared using the procedure in Example 2.140.13,replacing Example 2.140.12 with Example 2.141.3. MS (ESI) m/e 1948.8(M−H)⁻.

2.141.5N-({(3S,5S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-5-[(2-sulfoethoxy)methyl]pyrrolidin-1-yl}acetyl)-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.13,7]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-3-(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50-heptadecaoxatripentacontan-53-yl)phenyl}-L-alaninamide

The title compound was prepared using the procedure in Example 2.140.14,replacing Example 2.140.13 with Example 2.141.4. ¹H NMR (501 MHz,dimethyl sulfoxide-d₆) δ 6 12.87 (s. 1H), 9.84 (s, 1H), 8.18 (d, 1H),8.03 (dd, 2H), 7.78 (d, 1H), 7.61 (d, 1H), 7.52 (d, 1H), 7.45 (ddd, 4H),7.40-7.32 (m, 2H), 7.30 (s, 1H), 7.22 (d, 1H), 7.07 (s, 2H), 6.96 (d,1H), 5.01 (d, 2H), 4.95 (s, 2H), 4.64 (t, 1H), 4.38 (t, 1H), 4.24-4.12(m, 2H), 4.00 (d, 1H), 3.88 (t, 2H), 3.78 (t, 3H), 3.64 (ddt, 2H), 3.49(dd, 62H), 3.43-3.37 (m, 6H), 3.23 (s, 3H), 3.01 (t, 2H), 2.84-2.68 (m,1.5H), 2.63 (dd, 4H), 2.36 (d, 0.5H), 2.08 (d, 3H), 1.74 (t, 2H), 1.25(dt, 6H), 1.17-1.00 (m, 6H), 0.99-0.72 (m, 11H). MS (ESI) m/e 1153.0(M−2H)²⁻.

2.142 Synthesis of2-[({[2-(3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydriosoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl][(3S)-34-dihydroxybutyl]carbamoyl)oxy)methyl]-5-(3-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}propyl)phenylbeta-D-glucopyranosiduronic Acid (Synthon VY) 2.142.13-(1-((3-(2-((((4-(3-aminopropyl)-2-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)((S)-3,4-dihydroxybutyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicacid

Example 2.142.1 was prepared by substituting Example 2.136.4 for(9H-fluoren-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-1-oxobutan-2-yl)carbamateand substituting Example 1.85 for Example 1.2.9 in Example 2.49.1. MS(ESI) m/e 1217.3 (M+H)⁺.

2.142.22-[({[2-((3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.13,7]dec-1-yl}oxy)ethyl][(3S)-3,4-dihydroxybutyl]carbamoyl)oxy)methyl]-5-(3-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}propyl)phenylbeta-D-glucopyranosiduronic acid

Example 2.142.2 was prepared by substituting Example 2.142.1 for Example2.49.1 in Example 2.54. 1H NMR (400 MHz, dimethyl sulfoxide-d6) δ ppm8.14 (d, 1H), 8.03 (dt, 1H), 7.81-7.76 (m, 1H), 7.61 (dd, 1H), 7.53-7.41(m, 3H), 7.38-7.32 (m, 2H), 7.28 (s, 1H), 7.18 (d, 1H), 7.06 (d, 2H),6.97-6.92 (m, 2H), 6.85 (dd, 1H), 5.10 (q, 2H), 5.01 (d, 1H), 4.96 (s,2H), 3.48-3.18 (m, 12H), 3.06 (q, 2H), 3.00 (t, 2H), 2.08 (s, 3H),1.77-0.66 (m, 16H). MS (ESI) m/e 1352.5 (M−H)⁻.

2.143 Synthesis of1-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl]({[4-(4-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}butyl)-2-(beta-D-glucopyranuronosyloxy)benzyl]oxy}carbonyl)amino}-1,2-dideoxy-D-arabino-hexitol(Synthon WI) 2.143.13-(1-((3-(2-((((4-(4-aminobutyl)-2-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)((3R,4S,5R)-3,4,5,6-tetrahydroxyhexyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid

The title compound was prepared by substituting Example 1.77.2 forExample 1.25 and Example 2.124.5 for Example 2.97.7 in Example 2.97.8.MS (ESI) m/e 1291 (M+H)⁺, 1289 (M−H)⁻.

2.143.21-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.13,7]dec-1-yl}oxy)ethyl]({[4-(4-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}butyl)-2-(beta-D-glucopyranuronosyloxy)benzyl]oxy}carbonyl)amino}-1,2-dideoxy-D-arabino-hexitol

The title compound was prepared by substituting Example 2.143.1 forExample 2.49.1 in Example 2.54. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆)δ ppm 8.04 (d, 1H), 7.81 (d, 1H), 7.61 (d, 1H), 7.54-7.43 (m, 3H),7.41-7.35 (m 2H), 7.29 (s, 1H), 7.18 (m, 1H), 7.03 (s, 2H), 6.97 (d,1H), 6.93 (s, 1H), 6.86 (d, 1H), 5.18-5.05 (m, 3H), 5.03 (d, 1H), 4.97(s, 2H), 4.01 (s, 2H), 3.91 (d, 1H), 3.87 (t, 2H), 3.83 (m, 2H), 3.72(s, 2H), 3.67 (m, 2H), 3.59 (dd, 2H), 3.50-3.27 (m, 16H), 3.14 (d, 2H),3.04 (m, 4H), 2.09 (s, 3H), 1.68 (m, 2H), 1.52 (m, 2H), 1.44-1.31 (m,4H), 1.26-1.14 (m, 4H), 1.10 (m, 4H), 0.98 (q, 2H), 0.85 (m, 6H). MS(ESI) m/e 1428 (M+H)⁺, 1426 (M−H)⁻.

2.144 Synthesis of1-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](([4-(4-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}butyl)-2-(beta-D-glucopyranuronosyloxy)benzyl]oxy}carbonyl)amino)-1,2-dideoxy-D-erythro-pentitol(Synthon WK) 2.144.13-(1-((3-(2-((((4-(4-aminobutyl)-2-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)((3S,4R)-3,4,5-trihydroxypentyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid

The title compound was prepared by substituting Example 1.80 for Example1.25 and Example 2.124.5 for Example 2.97.7 in Example 2.97.8. MS (ESI)n/e 1261 (M+H)⁺, 1259 (M−H)⁻.

2.144.21-{[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.13,7]dec-1-yl}oxy)ethyl]({[4-(4-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}butyl)-2-(beta-D-glucopyranuronosyloxy)benzyl]oxy}carbonyl)amino}-1,2-dideoxy-D-erythro-pentitol

The title compound was prepared by substituting Example 2.144.1 forExample 2.49.1 in Example 2.54. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆)δ ppm 8.08 (t, 1H), 8.03 (d, 1H), 7.79 (d, 1H), 7.62 (d, 1H), 7.53-7.42(m, 3H), 7.38-7.33 (m, 2H), 7.20 (s, 1H), 7.17 (m, 1H), 7.07 (s, 2H),6.97-6.93 (m, 2H), 6.85 (d, 1H), 5.17-5.05 (m, 3H), 5.02 (d, 1H), 4.96(s, 2H), 3.98 (s, 2H), 3.88 (m, 4H), 3.80 (m 4H), 3.67 (m, 2H), 3.42 (m,4H), 3.36-3.23 (m, 13H), 3.08-2.99 (m, 5H), 2.09 (s, 3H), 1.86 (m, 1H),1.53 (m, 2H), 1.38 (m, 4H), 1.25 (m, 4H), 1.11 (m, 4H), 0.96 (m, 2H),0.83 (m, 6H). MS (ESI) m/e 1398 (M+H)⁺, 1396 (M−H)⁻.

2.145 Synthesis ofN-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-3-[27-(2,5,8,11,14,17,20,23-octaoxahexacosan-26-yl)-2,5,8,11,14,17,20,23-octaoxa-27-azatriacontan-30-yl]phenyl}-L-alaninamide(Synthon WP) 2.145.1 Tert-butyl((S)-1-(((S)-1-((3-(3-(((benzyloxy)carbonyl)amino)prop-1-yn-1-yl)-4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate

To a mixture of tert-butyl((S)-1-(((S)-1-((4-(hydroxymethyl)-3-iodophenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(0.5 g) in N,N-dimethylformamide (6 mL) was added benzylprop-2-yn-1-ylcarbamate (0.182 g). CuI (9.2 mg),bis(triphenylphosphine)palladium(II) dichloride (35 mg) andN,N-diisopropylethylamine (1.0 mL). The mixture was stirred at roomtemperature overnight. The mixture was concentrated under vacuum. Theresidue was dissolved in ethyl acetate (300 mL), washed with water,brine, dried over anhydrous sodium sulfate, filtered and concentrated.Evaporation of the solvent, and purification of the residue by silicagel chromatography, eluting with 30% ethyl acetate in dichloromethane,gave the title compound. MS (APCI) m/e 581.2 (M−H)⁻.

2.145.2 Tert-butyl((S)-1-(((S)-1-((3-(3-aminopropyl)-4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate

To a mixture of Example 2.145.1 (1.7 g) in ethanol (30 mL) was added 5%Pd/C (0.3 g) and cyclohexene (large excess). The reaction was stirred at100° C. for 45 minutes. The reaction was filtered and concentrated underreduced pressure. The residue was dissolved in N,N-dimethylformamide andpurified by reverse-phase HPLC on a Gilson system (C18 column), elutingwith 20-80% acetonitrile in water containing 0.1% trifluoroacetic acid,to give the title compound. MS (ESI) m/e 451.1 (M−H)⁻.

2.145.3 tert-butyl((S)-1-(((S)-1-((3-(27-(2,5,8,11,14,17,20,23-octaoxahexacosan-26-yl)-2,5,8,11,14,17,20,23-octaoxa-27-azatriacontan-30-yl)-4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate

To a mixture of Example 2.145.2 (45 mg) in dichloromethane (4 mL) wasadded 2,5,8,11,14,17,20,23-octaoxahexacosan-26-a (79 mg) followed byNaH(OAc)₃ (63.5 mg). The mixture was stirred at room temperature for 3hours and then concentrated under reduced pressure. The residue wasdissolved in N,N-dimethylformamide and purified by reverse-phase HPLC ona Gilson system (C18 column), eluting with 20-80% acetonitrile in watercontaining 0.1% trifluoroacetic acid, to give the title compound. MS(ESI) m/e 1212.1 (M−H)⁻.

2.145.4 tert-butyl((S)-1-(((S)-1-((3-(27-(2,5,8,11,14,17,20,23-octaoxahexacosan-26-yl)-2,5,8,11,14,17,20,23-octaoxa-27-azatriacontan-30-yl)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate

To a mixture of Example 2.145.3 (80 mg) in N,N-dimethylformamide (2 mL)was added bis(4-nitrophenyl) carbonate (26 mg) followed byN,N-diisopropylamine (0.012 mL). The mixture was stirred at roomtemperature overnight and purified directly by reverse phase HPLC on aGilson system (C18 column), eluting with 20-80% acetonitrile in watercontaining 0.1% trifluoroactic acid, to give the title compound. MS(ESI) m/e 1376.97 (M−H)⁻.

2.145.53-(1-((3-(2-((((2-(27-(2,5,8,11,14,17,20,23-octaoxahexacosan-26-yl)-2,5,8,11,14,17,20,23-octaoxa-27-azatriacontan-30-yl)-4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)(2-sulfoethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)naphthalen-2-yl)picolinicacid

To a mixture of Example 2.145.4 (30 mg) in N,N-dimethylformamide (4 mL)was added Example 1.43 (18.68 mg) followed by 1-hydroxybenzotriazolehydrate (3.4 mg) and N,N-diisopropylamine (3.84 uL). The mixture wasstirred at room temperature overnight. Trifluoroacetic acid (0.55 mL)was added to the mixture and stirred at room temperature for 3 hours.The mixture was purified by reverse-phase HPLC on a Gilson system (C18column), eluting with 20-80% acetonitrile in water containing 0.1%trifluoroacetic acid, to give the title compound. MS (ESI) me 1986.6(M−H)⁻.

2.145.6N-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)naphthalen-2-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-3-[27-(2,5,8,11,14,17,20,23-octaoxahexacosan-26-yl)-2,5,8,11,14,17,20,23-octaoxa-27-azatriacontan-30-y]phenyl}-L-alaninamide

The title compound was prepared as described in Example 2.123.21,replacing Example 2.123.20 with Example 2.145.5. ¹H NMR (400 MHz,dimethyl sulfoxide-d₆) δ ppm 13.10 (s, 1H), 9.92 (s, 1H), 9.43 (s, 1H),9.02 (s, 1H), 8.37 (dd, 1H), 8.30-8.14 (m, 5H), 8.07 (d, 1H), 8.02 (d,1H). 7.96 (d, 1H), 7.81 (d, 1H), 7.74-7.68 (m 1H), 7.57 (s, 1H),7.52-7.45 (m, 2H), 7.42-7.34 (m. 2H), 7.28 (d, 1H), 7.08 (s, 2H), 5.05(d, 2H), 4.39 (t, 1H), 4.21 (dd. 1H), 4.12 (s, 2H), 3.88 (s. 2H). 3.49(d, 55H), 3.34 (s, 200H), 3.23 (s. 5H), 3.13 (d, 4H), 2.79-2.65 (m, 5H),2.23 (s. 3H), 1.94 (d, 8H), 1.47-0.94 (m, 15H), 0.92-0.76 (m, 12H).

2.146 Synthesis of(6S)-2,6-anhydro-6-(2-{2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-5-({N-[(2S)-3-[3,4-bis(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yloxy)phenyl]-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]-L-valyl-L-alanyl}amino)phenyl}ethyl)-L-gulonicAcid (Synthon XD) 2.146.1(S)-2-(((benzyloxy)carbonyl)amino)-3-(3,4-dihydroxyphenyl)propanoic Acid

To a mixture of (S)-2-amino-3-(3,4-dihydroxyphenyl)propanoic acid (1.00kg) and NaHCO₃ (1.28 kg) in dioxane (5.00 L) and water (5.00 L) wasadded benzyl carbonochloridate (1.04 k) dropwise. The reaction mixturewas stirred at 25° C. for 12 hours. The reaction mixture was adjusted topH=3.0 4.0 by addition of 6 N aqueous HCl and extracted with ethylacetate (25 L). The organic layer was dried over Na₂SO₄, filtered, andconcentrated in vacuo to afford the title compound. ¹H NMR (400 MHz,dimethyl sulfoxide-d₆) δ ppm 8.73 (s, 1H), 7.54-7.26 (m, 8H), 6.64-6.45(m 3H), 4.98 (s, 2H), 4.49 (s. 1H), 2.87 (d, J=9.60 Hz. 1H), 2.68-2.62(m, 1H).

2.146.2 (S)-benzyl2-(((benzyloxy)carbonyl)amino)-3-(3,4-dihydroxyphenyl)propanoate

To a mixture of Example 2.146.1 (800.00 g) and Cs₂CO₃ (1.18 kg) wasadded bromoethylbenzene (259.67 g) at 20° C. The reaction mixture wasstirred for 1 hour, and TLC showed the reaction was complete. Theresidue was diluted with H₂O (5 L) and extracted with ethyl acetate(three times 5 L). The combined organic layers were washed with brine (5L), dried over Na₂SO₄ (150 g), filtered, and concentrated under reducepressure. The residue was purified by column chromatography (SiO₂,petroleum ether/ethyl acetate=100:1 to 1:1) twice to provide the titlecompound. ¹H NMR (400 MHz, CDCl₃) δ ppm 2.77-3.02 (m, 2H), 4.47 (br. s.,1H), 4.61 (d, J=7.94 Hz, 1H), 5.01-5.17 (m, 4H), 5.35-5.47 (m, 1H), 6.32(br. s., 1H), 6.38 (d. J=7.94 Hz, 1H), 6.51 (s, 1H), 6.65 (d, J=7.94 Hz,1H), 7.17-7.42 (m, 9H).

2.146.3 (S)-benzyl2-(((benzyloxy)carbonyl)amino)-3-(3,4-bis(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yloxy)phenyl)propanoate

To a mixture of K₂CO₃ (27.04 g) and K (5.95 g) in N,N-dimethylformamide(150 mL) was added Example 2.146.2 (8.12 g) and2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yl4-methylbenzenesulfonate (27.00 g) in dimethylformamide (150 mL). Themixture was stirred at 75° C. for 12 hours under N₂. Two additionalvials were set up as described above. All three reaction mixtures werecombined for purification. The mixture was poured into NH₄Cl aqueousmixture (9 L), and extracted with ethyl acetate (five times with 900mL). The combined organic layers were washed with brine (1500 mL), driedover Na₂SO₄ (150 g), filtered, and concentrated under reduce pressure toafford the crude residue. The residue was purified by columnchromatography (SiO₂, dichloromethan/methanol=100/1 to 20:1) to providethe title compound. ¹H NMR (400 MHz, CDCl₃) δ ppm 2.95-3.08 (m, 2H),3.38 (s, 6H), 3.57-3.68 (m, 80H), 3.78 (t, J=4.85 Hz, 2H), 3.83 (t,J=5.29 Hz, 2H), 4.01 (t, J=5.07 Hz. 2H), 4.10 (t, J=5.07 Hz, 2H),4.58-4.70 (m, 1H), 5.09 (s, 2H), 5.14 (d, J=3.53 Hz, 2H), 6.55 (d.J=8.38 Hz, 1H), 6.62 (d, J=1.76 Hz, 1H), 6.74 (d. J=7.94 Hz, 1H),7.27-7.49 (m, 10H).

2.146.4(S)-2-amino-3-(3,4-bis(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yloxy)phenyl)propanoicacid

To a mixture of Example 2.146.3 (16.50 g) in methanol (200 mL) was addedPd/C (9.00 g), and the mixture was stirred at 50° C. under H₂ (50 psi)for 16 hours. An additional reaction was set up as described above.LC/MS showed the reaction was complete, and both reaction mixtures werecombined for purification. The mixture was filtered and concentrated.The crude title compound was used in the next step without furtherpurification.

2.146,5(S)-3-(3,4-bis(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yloxy)phenyl)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoicacid

To a mixture of Example 2.146.4 (5.94 g) in H₂O (60.00 mL) was addedNa₂CO₃ (790.67 mg) and methyl 2,5-dioxypyrrole-1-carboxylate (1.19 g).The mixture was stirred at 25° C. for 3 hours. Four additional reactionswere set up as described above. All five reaction mixtures were combinedfor purification. Aqueous 4M HCl was added to adjust the pH to 2. Thecombined mixture was purified by preparatory reverse-phase HPLC(trifluoroacetic acid conditions) to provide the title compound. ¹H NMR(400 MHz, CDCl₃) δ ppm 3.35-3.40 (m, 6H), 3.51-3.58 (m, 4H), 3.58-3.75(m, 78H), 3.81 (q, J=4.70 Hz, 4H), 4.11 (dt, J=10.14, 5.07 Hz, 4H), 4.91(dd, J=11.47, 5.29 Hz, 1H), 6.53-6.69 (m, 3H), 6.71-6.89 (m, 2H). MS(ESI) m/e 638.0 (M+H)⁺.

2.146.6(6S)-2,6-anhydro-6-(2-{2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.13,7]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-5-({N-[(2S)-3-[3,4-bis(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yloxy)phenyl]-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]-L-valyl-L-alanyl}amino)phenyl}ethyl)-L-gulonicAcid

A mixture of Example 2.146.5 (0.020 mL),0-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (0.014 g) and N-ethyl-N-isopropylpropan-2-amine(0.020 mL) was stirred in N,N-dimethylformamide (0.4 mL) for 5 minutes.The mixture was added to a mixture of Example 2.123.20 (0.042 g) andN-ethyl-N-isopropylpropan-2-amine (0.020 mL) in N,N-dimethylformamide(0.4 mL) and it was stirred at room temperature for 3 hours. Thereaction was diluted with a mixture of water (1.5 mL),N,N-dimethylformamide (0.5 mL) and 2,2,2-trifluoroacetic acid (0.054 mL)and purified by preparatory reverse-phase HPLC on a Gilson 2020 system,using a gradient of 5% to 85% acetonitrile/water. The product-containingfractions were lyophilized to give the title compound. ¹H NMR (501 MHz,dimethyl sulfoxide-d₆) 12.86 (s. 4H), 9.92 (s, 2H), 8.26 (d, 1H), 8.10(s. 1H), 8.02 (dd, 1H), 7.77 (d, 1H), 7.64 (s. 1H), 7.54-7.49 (m, 1H),7.49-7.39 (m, 2H). 7.39-7.31 (m, 2H), 7.28 (s, 1H), 7.20 (d, 1H), 6.94(d, 1H), 6.87 (s, 2H), 6.77 (d, 1H), 6.60-6.53 (m, 1H), 5.05-4.91 (m5H), 4.80 (dd. 2H), 4.37 (t, 2H), 4.21 (t, 2H), 3.97 (dt. 3H), 3.86 (t,3H), 3.78 (d, 3H), 3.68 (dt, 4H), 3.65-3.28 (m, 102H), 3.20-3.08 (m,2H), 2.99 (t, 2H), 2.92 (d, 2H), 2.68 (dd. 2H), 2.07 (d, 4H), 1.54 (s,2H), 1.37-0.71 (m, 16H). MS (ESI) m/e 2631.2 (M−H)⁻.

2.147 Synthesis ofN-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-N-(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yl)-beta-alanyl-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-3-(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50-heptadecaoxatripentacontan-53-yl)phenyl}-L-alaninamide(Synthon XK) 2.147.1 Benzyl2,5,8,11,14,17,20,23,26,29,32-undecaoxa-35-azaoctatriacontan-38-oate

To a mixture of2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-amine (1 g) inN,N-dimethylformamide (4 mL) and water (3 mL) was added benzyl acrylate(0.377 g), dropwise. The reaction mixture was stirred overnight purifiedby reverse-phase HPLC on a Gilson system (C18 column), eluting with20-70% acetonitrile in water containing 0.1% trifluoroacetic acid, togive the title compound. MS (ESI) m/e 678.4 (M+H)⁺.

2.147.22,5,8,11,14,17,20,23,26,29,32-undecaoxa-35-azaoctatriacontan-38-oic Acid

Example 2.147.1 (220 mg) and 10% Pd/C (44 mg, dry) in tetrahydrofuran(10 mL) was shaken in a pressure bottle for 1 hour under 50 psi ofhydrogen gas. The reaction was filtered, and the filtrate wasconcentrated. The residue was dried under high vacuum to provide thetitle compound. MS (ESI) m/e 588.3 (M+H)⁺.

2.147.3 2,5-dioxopyrrolidin-1-yl35-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl)-2,5,8,11,14,17,20,23,26,29,32-undecaoxa-35-azaoctatriacontan-38-oate

A cold (0° C.) mixture of 2,5-dioxopyrrolidin-1-yl2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (566 mg),1-hydroxybenzotriazole hydrate (229 mg), 1-hydroxypyrrolidine-2,5-dione(86 mg) and Example 2.147.2 (440 mg) in N,N-dimethylformamide (3 mL) wastreated with N,N-diisopropylethylamine (785 μL) for 25 minutes. Thereaction was diluted with dimethyl sulfoxide and purified byreverse-phase HPLC on a Gilson system (C18 column), eluting with 5-55%acetonitrile in water containing 0.1% trifluoroacetic acid, to give thetitle compound. MS (ESI) m/e 822.3 (M+H)⁺.

2.147.4N-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-N-(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yl)-beta-alanyl-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.13,7]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-3-(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50-heptadecaoxatripentacontan-53-yl)phenyl}-L-alaninamide

To a cold (0° C.) mixture of Example 2.141.4 (28 mg), Example 2.147.3(27.1 mg) and 1-hydroxybenzotriazole hydrate (6.6 mg) inN,N-dimethylformamide (0.8 mL) was added N,N-diisopropylethylamine-2(20.1 μL). The mixture was stirred for 10 minutes and was purified byreverse-phase HPLC on a Gilson system (C18 column), eluting with30-70/acetonitrile in water containing 0.1% trifluoroacetic acid, togive the title compound. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ 12.81(s. 1H), 9.84 (s, 1H), 8.21-7.86 (m, 2H), 7.75 (d, 1H), 7.57 (d, 1H),7.52-7.28 (m, 7H), 7.27-7.15 (m, 2H), 7.04 (d, 2H), 6.91 (d, 1H), 4.94(d, 4H), 4.36 (dt, 3H), 4.19 (dt, 1H), 3.84 (t, 2H), 3.75 (d, 2H), 3.63(d, 1H), 3.46 (dd, 104H), 3.36 (s, 2H), 3.19 (s, 5H), 2.97 (t, 2H), 2.57(t, 5H), 2.42-2.26 (m, 1H), 2.03 (s, 7H), 2.00-1.83 (m, 1H), 1.70 (t,2H), 1.38-0.96 (m, 13H), 0.96-0.69 (m, 13H). MS (ESI) m/e 1327.7(M−2H)²⁻.

2.148 Synthesis ofN-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-N-(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yl)-beta-alanyl-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N-carbamoyl-L-ornithinamide(Synthon XL)

The title compound was prepared using the procedure in Example 2.147.4,replacing Example 2.141.4 with Example 2.112.2. ¹H NMR (400 MHz,dimethyl sulfoxide-d₆) δ 12.83 (s, 1H), 9.96 (d, 1H), 8.18-7.85 (m, 3H),7.75 (d, 1H), 7.64-7.37 (m, 7H), 7.32 (td. 2H), 7.28-7.20 (m. 3H), 7.04(s. 2H), 6.92 (d, 1H), 5.17-4.79 (m, 4H), 4.59-4.31 (m, 3H), 4.21 (dt,1H), 3.84 (t, 2H), 3.77 (d, 2H), 3.52 (s, 4H), 3.39 (d, 2H), 3.19 (s.5H), 2.94 (dt, 4H), 2.60 (t, 3H), 2.43-2.27 (m, 1H), 2.05 (s, 4H), 1.60(d, 2H), 1.44-0.57 (m, 22H). MS (ESI) m/e 1964.8 (M−H)⁻.

2.149 Synthesis ofN-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-3-[27-(2,5,8,11,14,17,20,23-octaoxahexacosan-26-yl)-2,5,8,11,14,17,20,23-octaoxa-27-azatriacontan-30-yl]phenyl}-L-alaninamide(Synthon YJ) 2.149.13-(1-((3-(2-((((2-(27-(2,5,8,11,14,17,20,23-octaoxahexacosan-26-yl)-2,5,8,11,14,17,20,23-octaoxa-27-azatriacontan-30-yl)-4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)(2-sulfoethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydrosoquinolin-2(1H)-yl)picolinicAcid

The title compound was prepared as described in Example 2.145.5,replacing Example 1.43 with Example 1.2.9. MS (ESI) m/e 1991.4 (M−H)⁻.

2.149.2N-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L-valyl-N-[4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.13,7]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-3-[27-(2,5,8,11,14,17,20,23-octaoxahexacosan-26-yl)-2,5,8,11,14,17,20,23-octaoxa-27-azatriacontan-30-yl]phenyl]-L-alaninamide

The title compound was prepared as described in Example 2.145, replacingExample 2.145.5 with Example 2.149.1. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 12.83 (s, 1H), 9.90 (s, 1H), 9.41 (s. 1H), 8.24 (d,2H), 8.01 (d, 1H), 7.77 (d, 1H), 7.67-7.29 (m, 8H), 7.26 (s. 2H), 7.06(s, 2H), 6.93 (d, 1H), 5.03 (d, 2H), 4.93 (s, 2H), 4.37 (t, 1H), 4.19(dd, 1H), 4.11 (s, 2H), 3.86 (t, 2H), 3.79 (s. 2H), 3.70-3.26 (m, 226H),3.21 (s. 6H), 3.11 (s, 5H), 2.99 (t, 2H), 2.66 (d, 4H), 2.08 (s. 3H).1.89 (s, 8H), 1.44-0.90 (m, 14H), 0.89-0.68 (m, 11H).

2.150 Synthesis of N-{(3S)-3-(2,5-dioxo-2,5-dihydro-1lH-pyrrol-1-yl)-3-[1-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-yl)-1H-1,2,3-triazol-4-yl]propanoyl}-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide(Synthon YQ) 2.150.13-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pent-4-ynoic Acid

To a mixture of 3-aminopent-4-ynoic acid trifluoroacetic acid salt (1.9g) in tetrahydrofuran (30 mL) was added methyl2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate (1.946 g), followed bythe rapid addition of N,N-diisopropylethylamine (8.04 mL). The resultingmixture was stirred at 60° C. for 16 hours. The mixture was concentratedto dryness. The residue was purified by reverse-phase HPLC on a Gilsonsystem (C18 column), eluting with 20-80% acetonitrile in watercontaining 0.1% trifluoroacetic acid, to give the title compound. MS(LC-MS) m/e 194 (M+H). ¹H-NMR (dimethyl sulfoxide-dc, 400 MHz) δ2.92-3.07 (m, 2H), 3.38 (d, 1H), 5.07-5.12 (m, 1H), 7.08 (s. 2H), 12.27(bs, 0.6H).

2.150.23-(1-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-yl)-1H-1,2,3-triazol-4-yl)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoicAcid

To Example 2.150.1 (700 mg) in a mixture of t-butanol/H₂O. (2:1, 15 mL)was added37-azido-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontane(2123 mg). Sodium(R)-2-((S)-1,2-dihydroxyethyl)-4-hydroxy-5-oxo-2,5-dihydrofuran-3-olate(71.8 mg) and copper(II) sulfate (28.9 mg) were sequentially added tothe mixture. The resulting mixture was stirred at room temperature for16 hours and concentrated. The residue was purified by reverse-phaseHPLC on a Gilson system (C18 column), eluting with 20-80/o acetonitrilein water containing 0.1% trifluoroacetic acid, to give the titlecompound. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ 3.24 (s, 3H),3.15-3.28 (m 2H). 3.41-3.52 (m, 44H), 3.79 (t, 2H), 4.48 (t, 2H),5.56-5.60 (m, 1H), 7.05 (s. 2H), 8.03 (s, 1H). MS (LC-MS) m/e 779(M+H)⁺.

2.150.3N-((3S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-[1-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-yl)-1H-1,2,3-triazol-4-yl]propanoyl-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.13,7]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N5-carbamoyl-L-ornithinamide

To a mixture of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (8.45 mg), and Example 2.150.2 (20 mg) inN,N-dimethylformamide (0.3 mL) at 0° C. was slowly addedN,N-diisopropylethylamine (22.19 μL), and the reaction mixture wasstirred for 1 minute. A cold (0° C.) mixture of Example 2.112.2 (20 mg)and N,N-diisopropylethylamine (22 sL) in N,N-dimethylformamide (0.4 mL)was added. The resulting mixture was stirred for 10 minutes and waspurified by reverse-phase HPLC on a Gilson system (C18 column), elutingwith 20-80% acetonitrile in water containing 0.1% trifluoroacetic acid,to give the title compound. (The absolute configuration of the3-position was arbitrarily assigned.) ¹H NMR (501 MHz, dimethylsulfoxide-d₆) δ 9.95 (s, 1H), 8.07 (d, 3H), 8.04-7.% (m, 2H), 7.77 (d,1H), 7.64-7.53 (m, 3H), 7.50 (s, 1H), 7.48-7.39 (m, 2H), 7.34 (q. 2H),7.30-7.23 (m, 3H), 6.98 (s, 2H), 6.93 (d, 1H), 5.61 (t, 1H), 4.96 (d,4H), 4.54-4.27 (m, 3H), 4.14 (t, 1H), 3.86 (t, 2H), 3.77 (q. 4H), 3.43(d, 71H), 3.21 (s, 6H), 3.00 (d, 5H), 2.61 (s, 2H), 2.07 (d, 3H), 1.92(s, 1H), 1.60 (d, 2H), 1.47-0.86 (m, 10H), 0.85-0.67 (m, 12H). MS (ESI)m/e 1010.6 (M−2H)²⁻.

2.151 Synthesis ofN-{(3R)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-[1-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-yl)-1H-1,2,3-triazol-4-yl]propanoyl}-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methy]phenyl}-N-carbamoyl-L-ornithinamide(Synthon YR)

Example 2.151 was isolated during the preparation of 2.150.3. (Theabsolute configuration of the 3-position was arbitrarily assigned.) ¹HNMR (501 MHz, dimethyl sulfoxide-d₆) δ 9.91 (s. 1H), 8.11 (dd, 2H),8.04-7.99 (m, 1H), 7.96 (s, 1H), 7.77 (d, 1H), 7.58 (t, 3H), 7.54-7.39(m, 2H), 7.39-7.31 (m, 2H), 7.31-7.24 (m, 3H), 7.00 (s, 2H), 6.94 (d,1H), 5.61 (dd, 1H), 5.08-4.79 (m, 4H), 4.40 (dt, 3H), 4.16 (s, 1f), 3.86(t, 2H), 3.82-3.73 (m, 4H), 3.51-3.30 (in, 46H), 3.21 6% (s, 7H),3.05-2.87 (m, 3H), 2.62 (t, 2H), 2.07 (d, 3H), 1.95 (s. 2H), 1.69 (s,1H), 1.51-0.86 (m, 10H), 0.88-0.70 (m, 13H). MS (ESI) m/e 1010.6(M−2H)²⁻.

2.152 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-[1-({3-[2-({[(2-{2-[(2S,3R,4R,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl]ethyl}-4-{[(2S)-2-{[(2S)-2-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl)oxy]carbonyl}[(3R,4S,5R)-3,4,5,6-tetrahydroxyhexyl]amino)ethoxy]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylicAcid (Synthon YS) 2.152.13-(1-((3-(2-((((4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)-2-(2-((2S,3R,4R,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)ethyl)benzyl)oxy)carbonyl)((3R,4S,5R)-3,4,5,6-tetrahydroxyhexyl)amino)ethoxy)-5,7-dimethyladamantan-1-y)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid

The title compound was prepared by substituting Example 1.77.2 forExample 1.25 and Example 2.123.19 for Example 2.97.7 in Example 2.97.8.MS (ESI) m/e 1417 (M+H), 1415 (M−H)⁺.

2.152.26-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-[1-({3-[2-({[(2-{2-[(2S,3R,4R,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl]ethyl}-4-{[(2S)-2-{([(2S)-2-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl)oxy]carbonyl}[(3R,4S,5R)-3,4,5,6-tetrahydroxyhexyl]amino)ethoxy]-5,7-dimethyltricyclo[3.3.1.13,7]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylicAcid

The title compound was prepared by substituting Example 2.152.1 forExample 2.49.1 in Example 2.54. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆)δ ppm 9.85 (m, 1H), 8.18 (t, 2H), 7.96 (d, 1H), 7.73 (d, 1H), 7.55 (d,1H), 7.46-7.25 (m, 8H), 7.21 (s. 1H), 7.15 (d, 1H), 7.00 (s, 1H), 6.99(d, 1H), 6.88 (d, 1H), 4.95 (bs, 2H), 4.88 (s, 2H), 4.32 (m, 1H), 4.15(t, 1H), 4.05 (s, 2H), 3.82 (t, 2H), 3.72 (m, 4H), 3.58-3.29 (m, 6H),3.19 (m, 4H), 3.11-3.00 (m, 6H), 2.97 (t, 2H), 2.91 (t, 2H), 2.72 (m,2H), 2.55 (m, 2H), 2.04 (s, 3H), 2.02-1.85 (m, 3H), 1.54 (m, 4H), 1.44(s, 1H), 1.33 (bs, 1H), 1.22 (m. 6H), 1.04 (m, 6H), 0.86 (m, 2H), 0.77(m, 12H). MS (ESI) m e 1554 (M+H)⁺, 1552 (M−H)⁻.

2.153 Synthesis of6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-[1-({3-[2-({[(2-{2-[(2S,3R,4R,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl]ethyl}-4-{[(2S)-2-({(2S)-2-[({(3S,5S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-5-[(2-sulfoethoxy)methyl]pyrrolidin-1-yl}acetyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl)oxy]carbonyl}[(3R,4S,5R)-3,4,5,6-tetrahydroxyhexyl]amino)ethoxy]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylicAcid (Synthon YY)

Example 2.119.15 (11 mg) was dissolved in N,N-dimethylformamide (0.1mL).2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisouroniumhexafluorophosphate(V) (11 mg) and N,N-diisopropylethylamine (7.4 mg)were added. The mixture was stirred at room temperature for fiveminutes. The mixture was then added to another mixture of Example2.152.1 (34 mg) and N,N-diisopropylethylamine (16.3 mg) inN,N-dimethylfornamide (0.2 mL). The reaction was stirred for 60 minutesat room temperature and quenched with trifluoroacetic acid (36 mg). Themixture was diluted with water (0.75 mL) and dimethyl sulfoxide (0.75mL) and purified by reverse-phase HPLC using 10-75% acetonitrile inwater (w/0.1% TFA) over 30 minutes on a Grace Reveleris equipped with aLuna column: C18(2), 100 A, 150×30 mm. Product fractions were pooled,frozen, and lyophilized to yield the title compound as thetrifluoroacetic acid salt. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm9.85 (m, 1H), 8.18 (d, 1H), 8.05 (d, 1H), 8.04 (d, 1H), 7.79 (d, 1H),7.53-7.39 (m, 8f), 7.36 (q, 2H), 7.29 (s, 1H), 7.22 (d, 1H), 7.07 (s,1), 6.96 (d, 1H), 5.18 (bs, 2H), 4.% (s, 2H), 4.65 (t, 1H), 4.37 (t,1H), 4.19 (t, 1H), 4.16 (s. 1H), 4.01 (d, 2H), 3.89 (t, 2H), 3.78 (m,4H), 3.73 (m. 2H), 3.49-3.44 (m, 4H), 3.40-3.20 (m, 8H), 3.24 (m, 4H),3.17-3.07 (m, 4H), 3.02 (t, 2H), 2.95 (t, 2H), 2.76 (m, 4H), 2.62 (m,1H), 2.37 (m, 1H), 2.09 (s, 3H), 1.99 (m, 2H), 1.86 (q, 1H), 1.62 (m,4H), 1.38 (bs, 2H), 1.28 (m, 6H), 1.18-1.02 (m, 6H), 0.96 (m, 2H),0.91-0.79 (m, 12H). MS (ESI) m/e 1773 (M−H)⁻.

2.154 Synthesis of(6S)-2,6-anhydro-6-(2-{2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-5-({N-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-N-(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yl)-beta-alanyl-L-valyl-L-alanyl}amino)phenyl}ethyl)-L-gulonicAcid (Synthon YT) 2.154.13-(1-((3-(2-((((4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)-2-(2-((2S,3R,4R,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)ethyl)benzyl)oxy)carbonyl)(2-sulfoethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid

A mixture of Example 1.2.9 (200 mg), Example 2.123.19 (288 mg), and1-hydroxybenzotriazole hydrate (50.2 mg) in N,N-dimethylformamide (2 mL)was cooled in an ice-bath, and N,N-diisopropylethylamine (143 μL) wasadded. The reaction mixture was stirred at room temperature for 2.5hours and concentrated. Tetrahydrofuran (0.5 mL) and methanol (0.5 mL)were added into the residue. The resulting mixture was cooled inice-bath and lithium hydroxide hydrate (147 mg) in water (2.5 mL) wasslowly added. The mixture was stirred at room temperature for 1.5 hours,and cooled in ice bath. Trifluoroacetic acid (361 μL) was added dropwiseuntil the pH reached 6. The mixture was purified by reverse-phase HPLCon a Gilson system (C18 column), eluting with 35-45% acetonitrile inwater containing 0.1% trifluoroacetic acid, to give the title compound.MS (ESI) m/e 1375.5 (M−H)⁻.

2.154.2(6S)-2,6-anhydro-6-(2-{2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.13,7]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-5-({N-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-N-(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yl)-beta-alanyl-L-valyl-L-alanyl}amino)phenyl}ethyl)-L-gulonicacid

To a mixture of 1-hydroxybenzotriazole hydrate (5.22 mg), Example2.154.1 (23.5 mg) and Example 2.147.3 (24 mg) in N,N-dimethylformamide(1 mL) at 0° C. was slowly added N,N-diisopropylethylamine (23.84 μL).The reaction mixture was stirred at room temperature for 15 minutes andpurified by reverse-phase HPLC on a Gilson system (C18 column), elutingwith 35-50% acetonitrile in water containing 0.1% trifluoroacetic acid,to give the title compound. ¹H NMR (501 MHz, dimethyl sulfoxide-d₆) δ12.83 (s, 1H), 9.88 (s, 1H), 8.23-8.04 (m, 2H), 8.02 (dd, 1H), 7.92 (s,1H), 7.77 (d, 1H), 7.59 (d, 1H), 7.55-7.30 (m, 7H), 7.27 (s, 1H), 7.20(d, 1H), 7.07 (d, 2H), 6.93 (d, 1H), 5.07-4.88 (m, 4H), 4.47-4.32 (m,3H), 4.22 (dt, 1H), 3.97-3.73 (m, 4H), 3.62-3.45 (m, 35H), 3.31 (t, 3H),3.21 (s, 3H), 3.06 (d, 2H), 2.83-2.54 (m, 5H), 2.47-2.29 (m, 1H),2.13-1.84 (m, 5H), 1.52 (d, 1H), 1.43-0.69 (m, 26H). MS (ESI) m/e 1043.0(M−2H)²⁻.

2.155 Synthesis of(6S)-2,6-anhydro-6-(2-{2-[({[2-(3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl)oxy)methyl]-5-[(N-{2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-[1-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-yl)-1H-1,2,3-triazol-4-yl]propanoyl}-L-valyl-L-alanyl)amino]phenyl}ethyl)-L-gulonicAcid (Synthon YU) 2.155.13-(1-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-yl)-1H-1,2,3-triazol-4-yl)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoicAcid

The title compound was prepared using the procedure in Example 2.150.2,replacing Example 2.150.1 with2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pent-4-ynoic acid.

2.155.2(6S)-2,6-anhydro-6-(2-{2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.13,7]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl)oxy)methyl]-5-[(N-12-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-[1-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-yl)-1H-1,2,3-triazol-4-yl]propanoyl}-L-valyl-L-alanyl)amino]phenyl}ethyl)-L-guonicAcid

The title compound was prepared using the procedure in Example 2.150.3,replacing Example 2.150.2 and Example 2.112.2 with Example 2.155.1 andExample 2.154.1, respectively. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ12.83 (s, 1H), 9.87 (d, 1H), 8.25-8.06 (m, 2H), 8.00 (d, 1H), 7.75 (d,1H), 7.71 (s. 1H), 7.57 (d, 1H), 7.54-7.28 (m, 6H), 7.25 (s, 1H), 7.18(d, 1H), 6.98-6.85 (m, 3H), 5.09-4.89 (m, 4H), 4.76 (ddd, 1H), 4.36(ddd, 3H), 4.17 (q, 1H), 3.84 (t, 2H), 3.76 (d, 2H), 3.72-3.66 (m, 2H),3.49-3.44 (m, 37H), 3.20 (s. 5H), 3.01-2.82 (m, 3H), 2.13-1.81 (m, 5H),1.52 (s. 1H), 1.39-0.50 (m, 23H). MS (ESI) m/e 1069.7 (M+2H)².

2.156 Synthesis of(6S)-2,6-anhydro-6-(2-{2-[({[2-(3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-5-[(N-{(3S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-[1-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-yl)-1H-1,2,3-triazol-4-yl]propanoyl-L-valyl-L-alanyl)amino]phenyl}ethyl)-L-gulonicAcid (Synthon YV)

Example 2.156 was isolated as a pure diastereomer during the preparationof Example 2.155.2. (The assignment of absolute configuration at the3-position is arbitrary.) ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) 12.82(s, 1H), 9.85 (s, 1H), 8.08 (d, 2H), 8.03-7.95 (m, 2H), 7.75 (d, 1H),7.57 (d, 1H), 7.51-7.29 (m, 6H), 7.24 (s, 1H), 7.18 (d, 1H), 6.95 (s.2H), 6.91 (d, 1H), 5.59 (dd, 1H), 5.06-4.86 (m, 4H), 4.43 (dt. 2H), 4.32(t, 1H), 4.11 (t, 1H), 3.84 (t, 2H), 3.75 (t, 3H), 3.55-3.41 (m, 43H),3.41-3.36 (m, 2H), 3.19 (s, 5H), 3.10 (t, 1H), 3.03-2.86 (m, 3H), 2.59(s, 3H), 2.13-1.82 (m, 6H), 1.52 (s, 1H), 1.37-0.65 (m, 26H). MS (ESI)m/e 1067.8 (M−2H)²⁻.

2.157 Synthesis of(6S)-2,6-anhydro-6-(2-{2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-5-[(N-{(3R)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-[1-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-yl)-1H-1,2,3-triazol-4-yl]propanoyl}-L-valyl-L-alanyl)amino]phenyl}ethyl)-L-gulonicAcid (Synthon YW)

Example 2.157 was isolated as a pure diastereomer during the preparationof Example 2.155.2. (The assignment of absolute configuration at the3-position is arbitrary.) ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) 12.81(s, 1H), 9.81 (s, 1H), 8.10 (d, 2H), 8.00 (d, 1H), 7.94 (s, 1H), 7.75(d, 1H), 7.57 (d, 1H), 7.51-7.28 (m, 6H), 7.24 (s, 1H), 7.18 (d, 1H),6.98 (s, 2H), 6.91 (d, 1H), 5.59 (t, 1H), 5.06-4.87 (m, 4H), 4.46-4.26(m, 2H), 4.12 (d, 1H), 3.84 (t, 2H), 3.75 (d, 3H), 3.46 (d. 27H),3.40-3.36 (m, 2H), 3.19 (s, 5H), 3.01-2.85 (m, 3H), 2.60 (s. 3H), 1.99(d, 4H), 1.52 (s, 1H), 1.35-0.65 (m, 23H). MS (ESI) m/e 1067.8 (M−2H)²⁻.

2.158 Synthesis of(6S)-2,6-anhydro-6-(2-{2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-5-[(N-{(3S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-[1-(3-sulfopropyl)-1H-1,2,3-triazol-4-yl]propanoyl}-L-valyl-L-alanyl)amino]phenyl}ethyl)-L-gulonicAcid (Synthon ZB) 2.158.1 Sodium 3-azidopropane-1-sulfonate

To a mixture of sodium azide (3.25 g) in water (25 mL) was added 1,2-oxathiolane 2,2-dioxide (6.1 g) in acetone (25 mL). The resultingmixture was stirred at room temperature for 24 hours and concentrated todryness. The solid was suspended in diethyl ether (100 mL) and stirredat reflux for 1 hour. The suspension was cooled to room temperature, andthe solid was collected by filtration, washed with acetone and diethylether, and dried under vacuum to afford the title compound. MS (LC-MS)m/e 164 (M−H)⁻.

2.158.2 Isopropyl 3-azidopropane-1-sulfonate

A mixture of Example 2.158.1 (6.8 g) in concentrated HCl (90 mL) wasstirred at room temperature for 1 hour. The mixture was concentrated todryness. The residue was dissolved in dichloromethane (350 mL), andtriisopropoxyethane (42.0 mL) was added in one portion to the mixture.The resulting mixture was stirred at 50° C. for 2 hours and concentratedto dryness. The crude residue was purified by silica gel chromatography,eluting with 10/1 petroleum ether/ethyl acetate, to give the titlecompound. ¹H-NMR (CDCl₃, 400 MHz): 1.42 (s, 3H), 1.44 (s, 3H), 2.08-2.15(m, 2H), 3.17 (t, 2H), 3.51 (t, 2H), 4.95-5.01 (m, 1H).

2.158.33-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-(1-(3-sulfopropyl)-1H-1,2,3-triazol-4-yl)propanoicAcid

To a mixture of Example 2.150.1 (450 mg) in r-butanol/H₂O (2:1, 9 mL)was added Example 2.158.2 (483 mg) followed by copper(II) sulfate (18.59mg) and sodium(R)-2-((S)-1,2-dihydroxyethyl)-4-hydroxy-5-oxo-2,5-dihydrofuran-3-olate(46.2 mg). The resulting mixture was stirred at room temperature for 16hours, and the mixture was concentrated to dryness. The residue waspurified by reverse-phase HPLC on a Gilson system (C18 column), elutingwith 20-80% acetonitrile in water containing 0.1% trifluoroacetic acid,to give the title compound. ¹H-NMR (dimethyl sulfoxide-d₆, 400 MHz):2.06-2.10 (m, 2H), 2.45-2.48 (m, 2H), 3.21-3.23 (m, 2H), 4.40-4.44 (m,2H), 5.55-5.59 (m, 1H), 7.05 (s, 2H), 8.10 (s, 1H). MS (LCMS) m/e 359(M+H)⁺.

2.158.4(6S)-2,6-anhydro-6-(2-{2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.13,7]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-5-[(N-{(3S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-[1-(3-sulfopropyl)-1H-1,2,3-triazol-4-yl]propanoyl}-L-valyl-L-alanyl)amino]phenyl}ethyl)-L-gulonicAcid

The title compound was prepared using the procedure in Example 2.150.3,replacing Example 2.150.2 and Example 2.112.2 with Example 2.158.3 andExample 2.154.1, respectively. The compound was isolated as a purediastereomer. (The absolute configuration of the 3-position wasarbitrarily assigned.) ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ10.14-9.66 (m, 1H), 8.07 (d, 2f), 8.04-7.% (m. 2H), 7.75 (d, 1H), 7.57(d, 1H), 7.52-7.29 (m, 7H), 7.26 (s. 1H), 7.18 (d, 1H). 6.92 (d, 3H),5.58 (t, 1H), 5.09-4.84 (m, 4H), 4.35 (dt, 3H), 4.15-4.02 (m, 1H),3.89-3.65 (m, 4H), 3.28 (d, 1H), 3.21 (dd, 2H), 3.14-3.02 (m, 2H),3.01-2.86 (m, 4H), 2.62 (d, 3H), 2.37 (t, 2H), 2.29 (s. OH), 2.02 (dt,5H), 1.52 (s, 1H), 1.40-0.59 (m, 24H). MS (ESI) m/e 1715.3 (M−H)⁻.

2.159 Synthesis of(6S)-2,6-anhydro-6-(2-{2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-5-[(N-{(3R)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-[1-(3-sulfopropyl)-1H-1,2,3-triazol-4-yl]propanoyl}-L-valyl-L-alanyl)amino]phenyl}ethyl)-L-gulonicAcid (Synthon ZC)

Example 2.159 was isolated as a pure diastereomer during the preparationof Example 2.158. (The absolute configuration of the 3-position wasarbitrarily assigned.) ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ 9.97(d, 1H), 8.21 (d, 1H), 8.13 (d, 1H), 8.04-7.96 (m, 2H), 7.75 (d, 1H),7.57 (d, 1H), 7.55-7.37 (m, 4H), 7.36-7.25 (m, 3H), 7.17 (d, 1H), 6.98(s, 2H), 6.93 (d, 1H), 5.58 (t, 1H), 4.94 (d, 4H), 4.50-4.26 (m, 3H),4.10 (s, 1H), 3.98-3.73 (m, 3H), 3.51 (d, 1H), 3.42 (s. 3H), 3.34-3.01(m, 6H), 3.01-2.83 (m, 4H), 2.63 (d, 4H), 2.42 (d, 1H), 2.18-1.80 (m,8H), 1.53 (s, 1H), 1.39-0.68 (m, 27H). MS (ESI) m/e 1715.4 (M−H)⁻.

2.160 Synthesis of(6S)-2,6-anhydro-6-(2-{2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-5-({N-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-N-[2-(2-sulfoethoxy)ethyl]-beta-alanyl-L-valyl-L-alanyl}amino)phenyl}ethyl)-L-gulonicAcid (Synthon ZJ) 2.160.14-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylbutyl2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethanesulfonate

To a mixture of tert-butyl (2-hydroxyethyl)carbamate (433 mg) indimethyl sulfoxide (0.9 mL) at 20° C. were added4-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylbutyl ethenesulfonate (500mg) and K₂CO₃ (210 mg). The mixture was warmed to 60° C., and stirredfor 16 hours in a capped bottle. The mixture was diluted with ethylacetate, washed with water and brine. The organic layer was dried overanhydrous sodium sulfate, filtered, and concentrated. The residue waspurified by silica gel flash chromatography, eluting with petrolether/ethyl acetate (10:1-2:1), to give the title compound. MS (LC-MS)m/e 630.3 (M+Na)⁺.

2.160.2 4-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylbutyl2-(2-amino)ethoxy)ethanesulfonate

To a mixture of Example 2.160.1 (1.5 g) in anhydrous dichloromethane(100 mL) at 20° C. was added zinc(II) bromide (0.445 g). The mixture wasstirred at room temperature for 16 hours.

Additional zinc(II) bromide (278 mg) was added to above mixture, and thereaction was stirred for additional 16 hours. The reaction was quenchedwith 1 M aqueous Na₂CO₃ mixture (5 mL), and the aqueous layer wasextracted with ethyl acetate three times. The combined organic layerswere dried over sodium sulfate, filtered, and concentrated. The residuewas purified by silica gel column chromatography, eluting withdichloromethane/methanol (10:1), to give the title compound. MS (LC-MS)m/e 508.2 (M+H)⁺.

2.160.3 Tert-butyl3-((2-(2-((4-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylbutoxy)sulfonyl)ethoxy)ethyl)amino)propanoate

To a mixture of Example 2.160.2 (0.365 g) in N, N-dimethylformamide (5.5mL) and water (0.55 mL) were added tert-butyl acrylate (0.105 mL) andtriethylamine (10.02 μL). The mixture was stirred at 60° C. for 30hours. The mixture was concentrated. The residue was mixed with 1 Maqueous Na₂CO₃ mixture (5 mL). The aqueous layer was extracted withethyl acetate three times. The combined organic layers were dried oversodium sulfate, filtered and concentrated. The residue was purified bysilica gel column chromatography, eluting with dichloromethane/ethylacetate (3:1) and dichloromethane/methanol (10:1), to give the titlecompound. MS (LC-MS) m/e 636.3 (M+H)⁺.

2.160.4 Tert-butyl3-(N-(2-(2-((4-((tert-butyldiphenysilyl)oxy)-2,2-dimethylbutoxy)sulfonyl)ethoxy)ethyl)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propanoate

To a mixture of Example 2.160.3 (557.5 mg),2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetic acid (272 mg) andO-(7-azabenzotriazol-1-yl)-N,N,N′.N′-tetramethyluroniumhexafluorophosphate (667 mg) in N, N-dimethylformamide (1.75 mL) at 0°C. was added N,N-diisopropylethylamine (0.459 mL). The resulting mixturewas stirred at 0° C. for 1 hour. The reaction mixture was mixed withsaturated aqueous NH₄Cl mixture, extracted with ethyl acetate and washedwith brine. The organic layer was dried over sodium sulfate, filteredand concentrated. The residue was purified by silica gel columnchromatography, eluting with petroleum ether/ethyl acetate (2/1), toprovide the title compound. MS (LC-MS) m/e 795.3 (M+Na)⁺.

2.160.53-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-(2-sulfoethoxy)ethyl)acetamido)propanoicAcid

To a mixture of Example 2.160.4 (230 mg) in dichloromethane (4 mL) wasadded trifluoroacetic acid (3 mL). The mixture was stirred at 20° C. for16 hours and was concentrated. The residue was purified by reverse-phaseHPLC on a Gilson system (C18 column), eluting with 20-80% acetonitrilein water containing 0.1% trifluoroacetic acid, to give the titlecompound. MS (LC-MS) m/e 379.0 (M+Na)⁺.

2.160.62-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(3-((2,5-dioxopyrrolidin-1-y)oxy)-3-oxopropyl)acetamido)ethoxy)ethane-1-sulfonicAcid

A mixture of 1-hydroxypyrrolidine-2,5-dione (16.43 mg), Example 2.160.5(30 mg). 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide hydrochloride(45.6 mg) in N,N-dimethylformamide were stirred overnight. The reactionmixture was purified by reverse-phase HPLC on a Gilson system (C18column), eluting with 2-30% acetonitrile in water containing 0.1%trifluoroacetic acid, to give the title compound. MS (ESI) m/e 475.9(M+H)⁺.

2.160.7(6S)-2,6-anhydro-6-(2-{2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.13,7]dec-1-yl}oxy)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]-5-({N-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-N-[2-(2-sulfoethoxy)ethyl]-beta-alanyl-L-valyl-L-alanyl}amino)phenyl}ethyl)-L-gulonicacid

To a mixture of 1-hydroxybenzotriazole hydrate (4.45 mg), Example2.160.6 (8.97 mg) and Example 2.154.1 (20 mg) in N,N-dimethylformamide(0.8 mL) at 0° C. was added N,N-diisopropylethylamine (20 μL dropwise).The reaction mixture was stirred at room temperature for 1 hour andpurified by reverse-phase HPLC on a Gilson system (C18 column), elutingwith 30-55% acetonitrile in water containing 0.1% trifluoroacetic acid,to give the title compound. ¹H NMR (500 MHz, dimethyl sulfoxide-d₆) δ12.87 (s, 1H), 9.88 (d, 1H), 8.28-8.10 (m, 1H), 8.03 (d, 1H), 7.95 (d.1H), 7.78 (d, 1H), 7.60 (d, 1H), 7.56-7.31 (m, 7H), 7.28 (s. 1H), 7.21(d, 1H), 7.06 (d, 2H), 6.95 (d, 1H), 5.06-4.90 (m, 4H), 4.38 (q. 3H),4.28-4.11 (m, 1H), 3.87 (t, 2H), 3.79 (d, 2H), 3.71-3.49 (m, 5H), 3.21(d, 2H), 3.12 (q, 2H), 2.97 (dt, 3H), 2.84-2.57 (m, 6H), 2.38 (dd, 1H),2.13-1.86 (m, 5H), 1.55 (s, 1H), 1.39-0.64 (m, 25H). MS (ESI) m/e 867.6(M−2H)².

2.161 Synthesis of6-{8-[(1,3-benzothiazol-2-yl)carbamoyl]-3,4-dihydroisoquinolin-2(1H)-yl}-3-[1-({3-[2-({[(2-{2-[(2S,3R,4R,5S,6S)-6-carboxy-3,4,5-trihydroxyoxan-2-yl]ethyl}-4-{[(2S)-2-{[(2S)-2-{[(2S)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-{4-[(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-y)oxy]phenyl}propanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}phenyl)methoxy]carbonyl}[(3R,4S,5R)-3,4,5,6-tetrahydroxyhexyl]amino)ethoxy]-5,7-dimethyltricyclo[3.3.1.1^(3,7)]decan-1-yl}methyl)-5-methyl-1H-pyrazol-4-yl]pyridine-2-carboxylicAcid (Synthon ZE)

The title compound was prepared by substituting Example 2.120.5 forExample 2.119.15 in Example 2.153. ¹H NMR (400 MHz, dimethylsulfoxide-d₆) δ ppm 12.84 (bs, 2H), 9.92 (m, 1H), 8.26 (d, 1H), 8.13 (d,1H), 8.03 (d, 1H), 7.79 (d, 1H), 7.61 (d, 1H), 7.52-7.41 (m, 4H), 7.36(m, 3H), 7.27 (s, 1H), 7.21 (d, 1H), 7.02 (d, 2H), 6.95 (d, 1H), 6.89(s, 2H), 6.78 (d, 2H), 5.02 (bs, 4H), 4.% (s. 2H), 4.59 (dd, 1H), 4.38(m, 2H), 4.21 (t, 1H), 3.99 (t, 2H), 3.88 (t, 2H), 3.79 (m, 2H), 3.69(t, 2H). 3.64 (m, 1H), 3.57 (m, 4H), 3.53 (m, 4H), 3.50 (s, 40H), 3.42(m, 2H), 3.38 (m, 1H), 3.30 (m, 2H), 3.23 (s, 6H), 3.20-3.08 (m, 6H),3.01 (t, 2H), 2.94 (t, 1H), 2.76 (m, 1H), 2.61 (m, 1H), 2.08 (s, 3H),2.06-1.92 (m, 2H), 1.67-1.52 (m, 3H), 1.38 (m, 1H), 1.32-1.22 (m, 6H),1.18-1.01 (m, 6H), 0.92 (m, 2H), 0.84 (m, 6H), 0.78 (m, 6H). MS (ESI)m/e 1078 (M−2H)-.

2.162 Synthesis of4-{[({2-[(3-{[4-(6-{8-[(1,3-benzothiazol-2-yl)carbamoyl]-3,4-dihydroisoquinolin-2(1H)-yl}-2-carboxypyridin-3-yl)-5-methyl-1H-pyrazol-1-yl]methyl}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]decan-1-yl)oxy]ethyl}[(3S)-3,4-dihydroxybutyl]carbamoyl)oxy]methyl}-3-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido]ethoxy}ethoxy)phenylbeta-D-glucopyranosiduronic Acid (Synthon ZS) 2.162.13-(1-((3-(2-((((2-(2-(2-aminoethoxy)ethoxy)-4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)(S)-3,4-dihydroxybutyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicacid

Example 2.162.1 was prepared by substituting Example 2.62.6 for(9H-fluoren-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)alnino)-1-oxo-5-ureidopentan-2-yl)amino)-1-oxobutan-2-yl)carbamateand substituting Example 1.85 for Example 1.2.9 in Example 2.49.1. MS(ESI) me 1261.4 (M−H)⁻.

2.162.24-{[({2-[(3-{[4-(6-{8-[(1,3-benzothiazol-2-yl)carbamoyl]-3,4-dihydroisoquinolin-2(1H)-yl}-2-carboxypyridin-3-yl)-5-methyl-1H-pyrazol-1-yl]methyl}-5,7-dimethyltricyclo[3.3.1.13,7]decan-1-yl)oxy]ethyl}[(3S)-3,4-dihydroxybutyl]carbamoyl)oxy]methyl}-3-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido]ethoxy}ethoxy)phenylbeta-D-glucopyranosiduronic acid

Example 2.162.2 was prepared by substituting Example 2.162.1 for Example2.49.1 in Example 2.54. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ 8.18(t, 1H), 8.00 (dd, 1H), 7.76 (d, 1H), 7.58 (dd, 1H), 7.50-7.29 (m, 6f),7.26 (s, 1H), 7.17 (d, 1H), 7.03 (s, 2), 6.92 (d, 1), 6.64 (d, 1), 6.57(dd. 1H), 4.94 (d, 4H), 4.08 (hept, 2H), 4.00 (s. 2H), 3.92-3.68 (m,8H), 3.51-3.13 (m, 12H), 2.98 (t, 2H), 2.06 (s, 3H), 1.65 (s, 1H),1.43-0.66 (m, 18H). MS (ESI) m/e 1398.5 (M−H)⁻.

2.163 Synthesis of2,6-anhydro-8-[2-({[{2-[(3-{[4-(6-{8-[(1,3-benzothiazol-2-yl)carbamoyl]-3,4-dihydroisoquinolin-2(1H)-yl}-2-carboxypyridin-3-yl)-5-methyl-1H-pyrazol-1-yl]methyl}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]decan-1-yl)oxy]ethyl}(2-sulfoethyl)carbamoyl]oxy}methyl)-5-{[(79S,82S)-74-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-82-methyl-77,80,83-trioxo-79-(propan-2-yl)-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-74,78,81-triazatrioctacontan-83-y]amino}phenyl]-7,8-dideoxy-L-glycero-L-gulo-octonicAcid (Synthon ZW) 2.163.1 benzyl2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-74-azaheptaheptacontan-77-oate

The title compound was prepared using the procedure in Example 2.147.1,replacing2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-amine with2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxatriheptacontan-73-amine.MS (ESI) m/e 625.9 (M+2H)².

2.163.2 2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-74-azaheptaheptacontan-77-oic Acid

The title compound was prepared using the procedure in Example 2.147.2,replacing Example 2.147.1 with Example 2.163.1. MS (ESI) m/e 1160.7(M+H)⁺.

2.163.3 2,5-dioxopyrrolidin-1-yl74-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl)-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-74-azaheptaheptacontan-77-oate

The title compound was prepared using the procedure in Example 2.147.3,replacing Example 2.147.2 with Example 2.163.2. MS (ESI) m/e 698.1(M+2H)⁺.

2.163.42,6-anhydro-8-[2-({[{2-[(3-[{4-(6-{8-[(1,3-benzothiazol-2-yl)carbamoyl]-3,4-dihydroisoquinolin-2(1H)-yl}-2-carboxypyridin-3-yl)-5-methyl-1H-pyrazol-1-yl]methyl}-5,7-dimethyltricyclo[3.3.1.13,7]decan-1-yl)oxy]ethyl}(2-sulfoethyl)carbamoyl]oxy}methyl)-5-{[(79S,82S)-74-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-82-methyl-77,80,83-trioxo-79-(propan-2-yl)-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-74,78,81-triazatrictacontan-83-y]amino}phenyl]-7,8-dideoxy-L-glycero-L-gulo-octonicAcid

The title compound was prepared using the procedure in Example 2.147.4,replacing Example 2.147.3 and Example 2.141.4 with Example 2.163.3 andExample 2.154.1, respectively. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ9.86 (s, 1H), 8.23-7.87 (m, 3H), 7.76 (d, 1H), 7.58 (dd, 1H), 7.53-7.25(m, 7H), 7.19 (d, 1H), 7.05 (d, 2H), 6.92 (d, 1H), 5.07-4.85 (m, 4H),4.49-4.30 (m, 3H), 4.20 (dt, 1H), 3.52 (d, 8H), 3.46-3.26 (m, 7H), 3.20(s, 4H), 3.15-2.82 (m, 4H), 2.61 (s, 3H), 2.38 (dq, 1H), 2.11-1.82 (m,5H), 1.53 (s, 1H), 1.39-0.66 (m, 24H). MS (ESI) m/e 1326.9 (M−2H)²⁻.

2.164 Synthesis of6-{8-[(1,3-benzothiazol-2-yl)carbamoyl]-3,4-dihydroisoquinolin-2(1H)-yl}-3-{1-[(3-{2-[{[(4-1{[(2S,5S)-2-[3-(carbamoylamino)propyl]-10-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-4,7-dioxo-5-(propan-2-yl)-15-sulfo-13-oxa-3,6,10-triazapentadecanan-1-oyl]amino}phenyl)methoxy]carbonyl}(2-sulfoethyl)amino]ethoxy}-5,7-dimethyltricyclo[3.3.1.13,7]decan-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicacid (Synthon ZX)

A mixture of 1-hydroxypyrrolidine-2,5-dione (2.74 mg),1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide hydrochloride (4.26 mg)and Example 2.160.5 (9.01 mg) in N,N-dimethylformamide (0.3 mL) werestirred at room temperature overnight. The mixture was cooled in icebath. 1-Hydroxybenaotriazole hydrate (3.65 mg) and a mixture of Example2.112.2 (20 mg) and N,N-diisopropylethylamine (22.19 μL) were added. Theresulting mixture was stirred at 0° C. for 10 minutes and purified byreverse phase HPLC, eluting with 30%-55% acetonitrile in 0.1%trifluoroacetic acid water, to provide the title compound. ¹H NMR (400MHz, dimethyl sulfoxide-d₆) δ 9.95 (d, 1H), 8.18-7.89 (m, 3H), 7.76 (d,1H), 7.57 (d, 3H), 7.52-7.21 (m, 8H), 7.04 (d, 2H), 6.92 (d, 1H), 4.94(d, 4H), 4.37 (d, 2H), 4.19 (d, 1H), 3.85 (t, 2H), 3.77 (d, 2H), 3.22(d, 2H), 2.96 (dt. 4H). 2.73 (dt, 2H), 2.66-2.55 (m, 2H), 2.36 (s, 1H),2.06 (s. 3H), 1.91 (s, 1H), 1.61 (d, 3H), 1.47-0.86 (m, 11H), 0.80 (ddd,12H). MS (ESI) m/e 1617.5 (M−H)⁻.

2.165 This Paragraph was Intentionally Left Blank 2.166 Synthesis of6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-(2-((((2-(2-((2S,3R,4R,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)ethyl)-4-((S)-2-((S)-2-(2-((3S,5S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-5-((2-sulfoethoxy)methyl)pyrrolidin-1-yl)acetamido)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)((S)-3,4-dihydroxybutyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinicAcid (Synthon AAA)

The title compound was prepared by substituting Example 2.167.1 forExample 2.119.16 in Example 2.119.17. ¹H NMR (500 MHz, dimethylsulfoxide-d₆) δ ppm 9.86 (br d, 1H), 8.17 (br d, 1H), 8.04 (m, 2H), 7.78(d, 1H), 7.61 (d, 1H), 7.51 (br d, 1H), 7.49-7.39 (m, 4H), 7.36 (m, 2H),7.29 (s, 1H), 7.21 (d, 1H), 7.07 (s, 2H), 6.95 (d, 1H), 5.00 (s. 2H),4.96 (s, 2H), 4.64 (t, 1H), 4.36 (m.I H). 4.19 (m, 1H), 4.16 (d, 1H),4.01 (d, 1H), 3.88 (br t, 2H), 3.82 (br m, 3H), 3.75 (br m, 1H), 3.64(t, 2H), 3.54 (d, 2H), 3.47 (m, 4H), 3.43 (br m, 4H), 3.23 (br m, 5H),3.13 (t, 1H), 3.10 (br m, 1H), 3.01 (br In, 2H), 2.93 (t, 1H), 2.83-2.68(m, 3H), 2.37 (m, 1H), 2.08 (s. 3H), 1.99 (br m, 2H), 1.85 (m, 1H), 1.55(br m, 1H), 1.37 (br m, 1H), 1.28 (br m, 6H), 1.10 (br m, 7H), 0.93 (brm, 1H), 0.88-.69 (m, 12H). MS (ESI) m/e 1713.6 (M−H)⁻.

Alternative Synthesis of6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-(2-((((2-(2-((2S,3R,4R,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)ethyl)-4-((S)-2-((S)-2-(2-((3S,5S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-5-((2-sulfoethoxy)methyl)pyrrolidin-1-yl)acetamido)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)((S)-3,4-dihydroxybutyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinicAcid (Synthon AAA) 2.166.13-(1-((3-(2-((((4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)-2-(2-((2S,3R,4R,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)ethyl)benzyl)oxy)carbonyl)((S)-3,4-dihydroxybutyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicAcid

To a stirred solution of Example 1.85 (0.065 g), 1-hydroxybenzotriazole(0.013 g) and N,N-diisopropylethylamine (0.06 mL) inN,N-dimethylformamide (0.5 mL) was added Example 2.123.19 (0.085 g), andthe mixture was stirred at room temperature for 2 hours. The reactionwas concentrated under reduced pressure. The residue was dissolved in asolvent mixture of methanol (0.5 mL) and tetrahydrofuran (0.5 mL), andlithium hydroxide monohydrate (30 mg) was added. The reaction wasstirred for 1 hour at ambient temperature, after which the reaction wasconcentrated under reduced pressure. The residue was dissolved inmethanol/water (1:1, 1 mL) containing 0.1 mL trifluoroacetic acid. Thesample was purified by reverse-phase HPLC (Phenomenex® Luna® C18 250×50mm column, 100 mL/min), eluting with 20-100% acetonitrile in watercontaining 0.01% trifluoroacetic acid over 40 minutes. The fractionscontaining product were lyophilized to give the title compound. MS (ESI)m/z 1357.5 (M+H)⁺.

2.166.26-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3-(1-((3-(2-((((2-(2-((2S,3R,4R,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)ethyl)-4-((S)-2-((S)-2-(2-((3S,5S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-5-((2-sulfoethoxy)methyl)pyrrolidin-1-yl)acetamido)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)((S)-3,4-dihydroxybutyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)picolinicAcid (Synthon AAA)

To a solution of Example 2.119.15 (16 mg) in N,N-dimethylformamide (200μL) was added1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate (16 mg. HATU) and N,N-diisopropylethylamine(17 μL). The reaction was stirred for 5 minutes, and a solution ofExample 2.166.1 (48 mg) and N,N-diisopropylethylamine (20 μL) inN,N-dimethylformamide (200 μL) was added. The reaction was stirred forone hour and diluted with a mixture of N,N-dimethylformamide/water (1/1,1.5 mL). The sample was purified by reverse-phase HPLC (Phenomenex®Luna® C18 250×50 mm column, 100 mL/min), eluting with 20-70%acetonitrile in water containing 0.01% trifluoroacetic acid over 40minutes. The fractions containing the product were lyophilized to givethe title compound. ¹H NMR (500 MHz, dimethyl sulfoxide-d₆) δ ppm 9.86(br d. 1H), 8.17 (br d, 1H), 8.04 (m, 2H), 7.78 (d, 1H), 7.61 (d, 1H),7.51 (br d, 1H), 7.49-7.39 (m, 4H), 7.36 (m, 2f), 7.29 (s. 1H), 7.21 (d,1H), 7.07 (s, 2H), 6.95 (d, 1f), 5.00 (s, 2H), 4.96 (s, 2H), 4.64 (t,1H), 4.36 (m, 1H), 4.19 (m, 1H), 4.16 (d, 1H), 4.01 (d, 1H), 3.88 (br t.2H), 3.82 (br m, 3H), 3.75 (br m, 1f). 3.64 (t, 2H), 3.54 (d, 2H), 3.47(m, 4H), 3.43 (br m, 4H), 3.23 (br m, 5H), 3.13 (t, 1H), 3.10 (br m,1H), 3.01 (br m, 2H), 2.93 (t, 1H), 2.83-2.68 (m, 3H), 2.37 (m, 1H),2.08 (s. 3H). 1.99 (br m, 2H), 1.85 (m, 1H), 1.55 (br m, 1H), 1.37 (brm, 1H), 1.28 (br m, 6H), 1.10 (br m, 7H), 0.93 (br m, 1H), 0.88-0.69 (m,12H). MS (ESI) m/z 1713.6 (M−H)⁻.

2.167 Synthesis of2,6-anhydro-8-(2-{[((2-[(3-{[4-(6-(8-[(1,3-benzothiazol-2-yl)carbamoyl]-3,4-dihydroisoquinolin-2(1H)-yl)-2-carboxypyridin-3-yl)-5-methyl-1H-pyrazol-1-yl]methyl}-5,7-dimethyltricyclo[3.3.1.13,7]decan-1-yl)oxy]ethyl)[(3S)-3,4-dihydroxybutyl]carbamoyl)oxy]methyl}-5-{[(2S)-2-({(2S)-2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido]-3-methylbutanoyl}amino)propanoyl]amino}phenyl)-7,8-dideoxy-L-glycero-L-gulo-octonicAcid (Synthon AAD) 2.167.13-(1-((3-(2-((((4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)-2-(2-((2S,3R,4R,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)ethyl)benzyl)oxy)carbonyl)((S)-3,4-dihydroxybutyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicacid

Example 2.167.1 was prepared by substituting Example 2.123.19 for(9H-fluoren-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-1-oxobutan-2-yl)carbamateand substituting Example 1.85 for Example 1.2.9 in Example 2.49.1. MS(ESI) m/e 1355.5 (M−H)⁻.

2.167.22,6-anhydro-8-(2-{[({2-[(3-{[4-(6-{8-[(1,3-benzothiazol-2-yl)carbamoyl]-3,4-dihydroisoquinolin-2(1H)-yl}-2-carboxypyridin-3-yl)-5-methyl-1H-pyrazol-1-yl]methyl}-5,7-dimethyltricyclo[3.3.1.13,7]decan-1-yl)oxy]ethyl}[(3S)-3,4-dihydroxybutyl]carbamoyl)oxy]methyl)-5-{[(2S)-2-(((2S)-2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido]-3-methylbutanoyl}amino)propanoyl]amino}phenyl)-7,8-dideoxy-L-glycero-L-gulo-octonicAcid

Example 2.167.2 was prepared by substituting Example 2.167.1 for Example2.49.1 in Example 2.54. ¹H NMR (501 MHz, dimethyl sulfoxide-d₆) δ 9.90(d, 1H), 8.25 (m, 2H), 8.01 (d, 1H). 7.77 (d, 1H), 7.59 (d, 1H),7.51-7.40 (m, 4H), 7.40-7.31 (m, 3H), 7.26 (s, 1H), 7.20 (d, 1H), 7.05(s. 2H), 6.93 (d, 1H), 4.96 (d, 4H), 4.36 (t, 1H), 4.22-4.06 (m, 3H),3.85 (t, 2H), 3.26-3.17 (m, 4H), 3.14-2.88 (m, 5H), 2.78-2.55 (m, 2H),2.10-1.88 (m, 5H), 1.69-1.49 (m, 2H), 1.39-0.73 (m, 28H). MS (ESI) m/e1492.5 (M−H)⁻.

2.168 Synthesis of2-{[({2-[(3-{[4-(6-{8-[(1,3-benzothiazol-2-yl)carbamoyl]-3,4-dihydroisoquinolin-2(1H)-yl}-2-carboxypyridin-3-yl)-5-methyl-1H-pyrazol-1-yl]methyl}-5,7-dimethyltricyclo[3.3.1.13,7]decan-1-yl)oxy]ethyl}[(3S)-3,4-dihydroxybutyl]carbamoyl)oxy]methyl}-5-{4-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido]butyl}phenylbeta-D-glucopyranosiduronic Acid (Synthon AAE) 2.168.13-(1-((3-(2-((((4-(4-aminobutyl)-2-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)((S)-3,4-dihydroxybutyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinicacid

Example 2.168.1 was prepared by substituting Example 2.124.5 for(9H-fluoren-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)anino)-1-oxo-5-ureidopentan-2-yl)amino)-1-oxobutan-2-yl)carbamateand substituting Example 1.85 for Example 1.2.9 in Example 2.49.1. MS(ESI) m/e 1229.5 (M−H)⁻.

2.168.22-{[({2-[(3-{[4-(6-{8-[(1,3-benzothiazol-2-yl)carbamoyl]-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl)-5-methyl-1H-pyrazol-1-yl]methyl}-5,7-dimethyltricyclo[3.3.1.13,7]decan-1-yl)oxy]ethyl}[(3S)-3,4-dihydroxybutyl]carbamoyl)oxy]methyl}-5-(4-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido]butyl}phenylbeta-D-glucopyranosiduronic Acid

Example 2.168.2 was prepared by substituting Example 2.168.1 for Example2.49.1 in Example 2.54. ¹H NMR (501 MHz, dimethyl sulfoxide-d₆) δ 8.07(s. 1H), 8.01 (dt, 1H), 7.77 (dt. 1H), 7.63-7.57 (m, 1H), 7.51-7.39 (m,3H), 7.38-7.31 (m, 2H), 7.26 (s, 1H), 7.16 (d, I1H), 7.05 (s, 2H), 6.93(d, 2H), 6.84-6.80 (m, 1H), 5.14-4.98 (m, 3H), 4.94 (s. 2H), 3.79 (d,2H), 3.48-3.19 (m, 10H), 3.08-2.96 (m, 4H), 2.52 (s, 4H), 2.07 (s, 2H),1.77-0.72 (m, 14H). MS (ESI) m/e 1366.5 (M−H)⁻.

2.169 Synthesis of6-{8-[(1,3-benzothiazol-2-yl)carbamoyl]-3,4-dihydroisoquinolin-2(H)-yl}-3-{1-[(3-{2-[{[(4-{[(2S)-5-(carbamoylamino)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}pentanoyl]amino}phenyl)methoxy]carbonyl}(2-sulfoethyl)amino]acetamido}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]decan-1-yl)methyl]-5-methyl-1H-pyrazol-4-yl}pyridine-2-carboxylicAcid (Synthon ABG)

The title compound was prepared as described in Example 2.54, replacingExample 2.49.1 with Example 1.89.12. ¹H NMR (501 MHz, dimethylsulfoxide-d₆) δ ppm 9.95 (d, 1H), 8.10-7.96 (m, 1H), 7.75 (t, 2H), 7.57(dd, 3H), 7.51-7.18 (m, 8H), 6.95 (d, 3H), 6.92 (s, 0H), 5.03-4.86 (m,4H), 4.36 (d, 1H), 3.85 (t, 2H), 3.78-3.67 (m, 4H), 3.42 (s. 2H), 3.33(t, 2H), 3.04-2.86 (m, 4H), 2.63 (d, 2H), 2.13 (dd, 1H), 2.07 (s, 3H),1.98-1.87 (m, OH), 1.71-1.23 (m, 10H), 1.24-0.85 (m, 6H), 0.78 (t, 1H).MS (ESI) m/e 1463.5 (M−H)⁻.

2.170 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.13,7]dec-1-yl}sulfanyl)ethyl](2-sulfoethyl)carbamoyl}oxy)methyl]phenyl}-N5-carbamoyl-L-ornithinamide(Synthon ABL)

The title compound was prepared by substituting Example 1.90.11 forExample 1.2.9 in Example 2.1. ¹H NMR (500 MHz, dimethyl sulfoxide-d₆) δppm 10.0 (s, 1H), 8.08 (br s, 1H), 8.03 (d, 1H), 7.81 (br s, 1H) 7.78(d, 1H), 7.60 (m, 3H) 7.52 (t, 1H), 7.47 (t, 1H), 7.43 (d, 1H), 7.37 (d,1H), 7.34 (d, 1H) 7.32 (s, 1H), 7.28 (d, 2H), 6.99 (s, 1H), 6.96 (d,2H), 5.00 (s, 2H), 4.96 (s, 2H), 4.39 (m, 1H), 4.18 (m, 2H), 3.88 (m,2H), 3.82 (s, 1H), 3.77 (s, 1H), 3.46 (br m, 2H), 3.58 (t, 2H), 3.29 (vbr m, 2H), 3.01 (br m, 3H), 2.95 (br m, 1H), 2.47 (m, 2f), 2.61 (br m,2H) 2.16 (m, 1H), 2.10 (m, 4H), 1.96 (br m, 1H), 1.69 (v br m. 1H), 1.59(v br m, 1H), 1.53-1.40 (m, 7H), 1.39-1.22 (m, 5H). 1.17 (m, 3H),1.13-0.88 (m, 6H), 0.87-0.77 (m, 9H), 0.75 (s, 3H). MS (ESI) m/e 1466.5(M−H)⁻.

2.171 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-[4-({[(3-{3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.13,7]dec-1-yl}propyl)(2-sulfoethyl)carbamoyl]oxy}methyl)phenyl]-N5-carbamoyl-L-ornithinamide(Synthon ABN)

The title compound was prepared as described in Example 2.1, replacingExample 1.2.9 with Example 1.91.13. ¹H NMR (501 MHz, DMSO-d₆) δ ppm12.83 (s. 1H), 9.96 (s, 1H), 8.03 (t, 2H), 7.77 (d, 2H), 7.64-7.52 (m,3H), 7.45 (ddd, 3H), 7.34 (td, 2H), 7.29-7.21 (m, 3H), 7.03-6.91 (m,3H), 4.95 (d, 4H), 4.37 (q, 1H), 4.17 (s. 1H), 3.86 (t, 2H), 3.45-3.29(m, 4H), 3.10 (t 2H), 2.95 (dt, 4H), 2.61 (q, 2H), 2.15 (td, 2H), 2.07(s, 3H), 2.00-1.89 (m, 1H), 1.74-1.24 (m, 10H), 1.25-0.87 (m, 13H),0.88-0.70 (m, 12H). MS (ESI) me 1450.2 (M+H)⁺.

2.172 Synthesis of2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.13,7]dec-1-yl}oxy)ethyl][(3S)-3,4-dihydroxybutyl]carbamoyl)oxy)methyl]-5-{4-[((3S,5S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-5-[(2-sulfoethoxy)methyl]pyrrolidin-1-yl}acetyl)amino]butyl}phenylbeta-D-glucopyranosiduronic Acid (Synthon AAF)

The title compound was prepared as described in Example 2.119.17,replacing Example 2.168.1 for Example 2.119.16. ¹H NMR (400 MHz,dimethyl sulfoxide-d₆) δ ppm 8.03 (d, 1H), 7.84 (br t. 1H), 7.78 (d,1H), 7.61 (d, 1H), 7.50 (br d, 1H), 7.45 (dd, 1H), 7.43 (d, 1H), 7.36(m, 2H), 7.29 (s, 1H), 7.17 (br m, 1H), 7.06 (s. 2H), 6.95 (m, 2H), 6.85(d, 1H), 5.08 (s. 2H), 5.02 (d, 1H), 4.96 (s, 2H), 4.70 (t, 1H), 4.06(d, 2H), 3.88 (m, 4H), 3.81 (m, 2H), 3.73 (br m, 1H), 3.62 (m, 2H), 3.47(br m, 4H), 3.40 (m, 4H), 3.35 (m, 2H), 3.29 (m, 4H), 3.07 (m, 2H), 3.00(t, 2H), 2.73 (m, 2H), 2.54 (m, 2H), 2.36 (br m, 1H), 2.09 (s, 3H), 1.83(m, 1H), 1.71 (br m, 1H), 1.55 (br m, 2H), 1.40 (br m, 5H), 1.24 (br m.4H), 1.10 (br m, 5H), 0.94 (br m, 1H), 0.83, 0.81 (both s, total 6H). MS(ESI) me 1587.5 (M−H)⁻.

2.173 Synthesis of2,6-anhydro-8-[2-({[{2-[(3-{[4-(6-{8-[(1,3-benzothiazol-2-yl)carbamoyl]-3,4-dihydroisoquinolin-2(1H)-yl}-2-carboxypyridin-3-yl)-5-methyl-1H-pyrazol-1-yl]methyl}-5,7-dimethyltricyclo[3.3.1.13,7]decan-1-yl)oxy]ethyl}(2-sulfoethyl)carbamoyl]oxy}methyl)-5-{[N-({(3R,5S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-5-[(2-sulfoethoxy)methyl]pyrrolidin-1-yl}acetyl)-L-valyl-L-alanyl]amino}phenyl]-7,8-dideoxy-L-glycero-L-gulo-octonicacid (Synthon ABO) 2.173.1(3R,6R,7aS)-6-azido-3-phenyltetrahydropyrrolo[1,2-c]oxazol-5(3H)-one

The title compound was prepared by substituting Example 2.119.3 forExample 2.119.2 in Example 2.119.4. MS (DCI) m/e 262.0 (M+NH₄)⁺.

2.173.2(3R,6R,7aS)-6-amino-3-phenyltetrahydropyrrolo[1,2-c]oxazol-5(3H)-one

The title compound was prepared by substituting Example 2.173.1 forExample 2.119.4 in Example 2.119.5. MS (DCI) m/e 219.0 (M+H)⁺.

2173.3(3R,6R,7aS)-6-(dibenzylamino)-3-phenytetrahydropyrrolo[1,2-c]oxazol-5(3H)-one

The title compound was prepared by substituting Example 2.173.2 forExample 2.119.5 in Example 2.119.6. MS (DCI) m/e 399.1 (M+H)⁺.

2.173.4 (3R,5S)-3-(dibenzylamino)-5-(hydroxymethyl)pyrrolidin-2-one

The title compound was prepared by substituting Example 2.173.3 forExample 2.119.6 in Example 2.119.7, with the exception that the reactionwas heated to 65° C. for one day rather than 6 days. MS (DCI) m/e 311.1(M+H)+.

2.173.5(3R,5S)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3-(dibenzylamino)pyrrolidin-2-one

The title compound was prepared by substituting Example 2.173.4 forExample 2.119.7 in Example 2.119.8. The title compound was carried on tothe next step without purification. MS (DCI) m/e 425.2 (M+H)⁺.

2.173.6 Tert-butyl2-((3R,5S)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3-(dibenzylamino)-2-oxopyrrolidin-1-yl)acetate

The title compound was prepared by substituting Example 2.173.5 forExample 2.119.8 in Example 2.119.9. The title compound was carried on tothe next step without purification. MS (DCI) m/e 539.3 (M+H)⁺.

2.173.7 Tert-butyl2-((3R,5S)-3-(dibenzylamino)-5-(hydroxymethyl)-2-oxopyrrolidin-1-yl)acetate

The title compound was prepared by substituting Example 2.173.6 forExample 2.119.9 in Example 2.119.10. MS (DCI) m/e 425.2 (M+H)⁺.

2.173.8 Tert-butyl2-((3R,5S)-5-((2-((4-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylbutoxy)sulfonyl)ethoxy)methyl)-3-(dibenzylamino)-2-oxopyrrolidin-1-yl)acetate

The title compound was prepared by substituting Example 2.173.7 forExample 2.119.10 in Example 2.119.11.

2.173.9 tert-butyl(S)-2-(2-((2-((4-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylbutoxy)sulfonyl)ethoxy)methyl)-5-oxopyrrolidin-1-yl)acetate

The title compound was prepared by substituting Example 2.173.8 forExample 2.119.11 in Example 2.119.12. MS (ESI) m/e 691.1 (M+H)⁺.

2.173.104-(((3R,5S)-1-(2-(tert-butoxy)-2-oxoethyl)-5-((2-((4-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylbutoxy)sulfonyl)ethoxy)methyl)-2-oxopyrrolidin-3-yl)amino)-4-oxobut-2-enoicAcid

The title compound was prepared by substituting Example 2.173.9 forExample 2.119.12 in Example 2.119.13. MS (ESI) m/e 789.0 (M+H)⁺.

2.173.11 Tert-butyl2-((3R,S)-5-((2-((4-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylbutoxy)sulfonyl)ethoxy)methyl)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxopyrrolidin-1-yl)acetate

The title compound was prepared by substituting Example 2173.10 forExample 2.119.13 in Example 2.119.14.

2.173.122-((3R,S5)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-5-((2-sulfoethoxy)methyl)pyrrolidin-1-yl)aceticAcid

The title compound was prepared by substituting Example 2.173.11 forExample 2.119.14 in Example 2.119.15. MS (ESI) m/e 377.0 (M+H)⁺.

2.173.132,6-anhydro-8-[2-({[{2-[(3-{[4-(6-{8-[(1,3-benzothiazol-2-yl)carbamoyl]-3,4-dihydroisoquinolin-2(1H)-yl}-2-carboxypyridin-3-yl)-5-methyl-1H-pyrazol-1-yl]methyl}-5,7-dimethyltricyclo[3.3.1.13,7]decan-1-y)oxy]ethyl}(2-sulfoethyl)carbamoyl]oxy}methyl)-5-{[N-({(3R,5S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-5-[(2-sulfoethoxy)methyl]pyrrolidin-1-yl}acetyl)-L-valyl-L-alanyl]amino}phenyl]-7,8-dideoxy-L-glycero-L-gulo-octonicAcid

The title compound was prepared by substituting Example 2.123.20 forExample 2.119.16 and Example 2.173.12 for Example 2.119.15 in Example2.119.17. ¹H NMR (400 MHz, dimethyl sulfoxide-d₆) δ ppm 9.94 (d, 1H),8.28 (br d, 1H), 8.01 (d, 2H), 7.77 (d, 1H), 7.59 (d, 1H), 7.53 (d, 1H),7.43 (m, 4H), 7.34 (m, 3H), 7.19 (d, 1H), 7.06 (s, 2H), 6.96 (d, 1H),4.99 (m, 2H), 4.95 (s, 2H), 4.78 (t, 1H), 4.36 (t, 1H), 4.19 (br m, 1H),4.16 (d, 1H), 3.98 (d, 1H), 3.87 (br t, 2H), 3.81 (br d, 2H), 3.73 (brm,1H), 3.63 (t, 2H), 3.53 (m, 2H), 3.44 (m, 4H), 3.31 (t, 2H), 3.21 (br m,2H), 3.17 (m, 2H), 3.00 (m, 2H), 2.92 (br m, I1H), 2.75 (m, 3H), 2.65(br m, 3H), 2.35 (br m, 1H), 2.16 (m, 1H), 2.07 (s, 3H), 1.98 (br m,2H), 1.55 (br m, 1H), 1.34 (br m, 1H), 1.26 (br m, 6H), 1.09 (br m, 7H),0.93 (br m, 1H), 0.87, 0.83, 0.79 (all d, total 12H). MS (ESI) m/e1733.3 (M−H)⁻.

2.174 Synthesis of2,6-anhydro-8-{2-({[{2-[(3-{[4-(6-{8-[(1,3-benzothiazol-2-yl)carbamoyl]-3,4-dihydroisoquinolin-2(1H)-yl}-2-carboxypyridin-3-yl)-5-methyl-1H-pyrazol-1-yl]methyl}-5,7-dimethyltricyclo[3.3.1.13,7]decan-1-yl)oxy]ethyl}(2-sulfoethyl)carbamoyl]oxy}methyl)-5-[(N-{[(3R,5S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-5-(41-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38-tridecaoxa-42-azatritetracontan-43-yl)pyrrolidin-1-yl]acetyl}-L-valyl-L-alanyl)amino]phenyl}-7,8-dideoxy-L-glycero-L-gulo-octonicAcid (Synthon ABM) 2.174.1 Tert-butyl[(3R,5S)-5-{[bis(tert-butoxycarbonyl)amino]methyl}-3-(dibenzylamino)-2-oxopyrrolidin-1-yl]acetate

To a cold (0° C.) solution of Example 2.173.7 (1.6 g) in dichloromethane(15 mL) was added triethylamine (0.70 mL) and methanesulfonyl chloride(0.39 mL) dropwise. The ice-bath was removed, and the reaction wasstirred at room temperature for two hours. The reaction was quenched bythe addition of saturated aqueous sodium bicarbonate solution. Thelayers were separated, and the organic layer was washed with brine. Thecombined aqueous layers were back-extracted with dichloromethane. Thecombined organic layers were dried with anhydrous sodium sulfate,filtered and concentrated under reduced pressure to give theintermediate mesylate (1.9 g). The residue was dissolved in acetonitrile(15 mL), and di-tert-butyl-iminodicarboxylate (1.0 g) and cesiumcarbonate (2.4 g) were added. The reaction was heated to reflux undernitrogen for one day. The reaction was cooled and quenched by theaddition of water and diethyl ether. The layers were separated, and theorganic was washed with brine. The combined aqueous layers wereback-extracted with diethyl ether. The combined organic layers weredried with anhydrous sodium sulfate, filtered and concentrated underreduced pressure. The residue was purified by silica gel chromatography,eluting with 20% ethyl acetate in heptanes, to give the title compound.MS (DCI) m/e 624.3 (M+H)⁺.

2.174.2 Tert-butyl [(3R,5S)-3-amino-5-{[bis(tert-butoxycarbonyl)amino]methyl}-2-oxopyrrolidin-1-yl]acetate

To a solution of Example 2.174.1 (1.0 g) in ethyl acetate (6 mL) andmethanol (18 mL) was added palladium hydroxide on carbon (100 mg, 20% byweight). The reaction was stirred at room temperature under a hydrogenballoon for one day. The reaction was filtered through diatomaceousearth, eluting with ethyl acetate. The filtrate was concentrated underreduced pressure, dissolved in dichloromethane (10 mL) and filteredthrough a syringe-tip Teflon 40 micron filter. The filtrate wasconcentrated under reduced pressure to give the title compound. MS (DCI)m/e 444.1 (M+H)⁺.

2.174.34-{[(3R,5S)-5-{[bis(tert-butoxycarbonyl)amino]methyl}-1-(2-tert-butoxy-2-oxoethyl)-2-oxopyrrolidin-3-yl]amino}-4-oxobut-2-enoicAcid

The title compound was prepared by substituting Example 2.174.2 forExample 2.119.12 in Example 2.119.13. MS (ESI) m/e 540.2 (M−H)⁻.

2.174.4 Tert-butyl[(3R,5S)-5-{[bis(tert-butoxycarbonyl)amino]methyl}-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxopyrrolidin-1-yl]acetate

The title compound was prepared by substituting Example 2.174.3 forExample 2.119.13 in Example 2.119.14. MS (DCI) me 541.1 (M+NH₄)⁺.

2.174.52-((3R,5S)-5-(aminomethyl)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxopyrrolidin-1-yl)aceticAcid

To a solution of Example 2.174.4 (284 mg) in dichloromethane (10 mL) wasadded trifluoroacetic acid (5 mL). The reaction was stirred at roomtemperature for two hours and was concentrated under reduced pressure.The residue was dissolved in water/acetonitrile 7/3 (5 mL), frozen andlyophilized to provide the title compound, which was used in thesubsequent step without further purification. MS (ESI) m/e 266.1 (M−H)⁻.

2.174.62-((3R,5S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-5-(41-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38-tridecaoxa-42-azatritetracontan-43-yl)pyrrolidin-1-yl)aceticAcid

To a solution of2,5,8,11,14,17,20,23,26,29,32,35,38-tridecaoxahentetracontan-41-oic acid(160 mg) in N,N-dimethylformamide (1.0 mL) was addedO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (85 mg) and N,N-diisopropylethylamine (130 L). Thereaction mixture was stirred for three minutes at room temperature, anda solution of Example 2.174.5 (70 mg) and N,N-diisopropylethylamine (130L) in N,N-dimethylformamide (1.0 mL) was added.

The reaction was stirred at room temperature for one hour and dilutedwith N,N-dimethylformamide/water 1/1 (3.5 mL). The solution was purifiedby reverse phase HPLC on a Gilson system (C18 column), eluting with20-70% acetonitrile in 0.1% TFA water, to provide the title compound. MS(ESI) m/e 880.4 (M−H)⁻.

2.174.72,6-anhydro-8-{2-({[{2-[(3-{[4-(6-{8-[(1,3-benzothiazol-2-yl)carbamoyl]-3,4-dihydroisoquinolin-2(1H)-yl}-2-carboxypyridin-3-yl)-5-methyl-1H-pyrazol-1-yl]methyl}-5,7-dimethyltricyclo[3.3.1.1^(3,7)]decan-1-yl)oxy]ethyl}(2-sulfoethyl)carbamoyl]oxy}methyl)-5-[(N-{[(3R,S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-5-(41-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38-tridecaoxa-42-azatritetracontan-43-yl)pyrrolidin-1-yl]acetyl}-L-valy-L-alanyl)amino]phenyl}-7,8-dideoxy-L-giycero-L-gulo-octonicAcid

The title compound was prepared by substituting Example 2.174.6 forExample 2.119.15 and Example 2.123.20 for Example 2.119.16 in Example2.119.17 ¹H NMR (500 MHz, dimethyl sulfoxide-d₆) δ ppm 9.93 (br d, 1H),8.28 (d, 1H), 8.03 (d, 1H), 8.02 (br s, 1H), 7.91 (br d, 1H), 7.79 (d,1H), 7.61 (d, 1H), 7.51 (br d, 1H), 7.49-7.42 (m, 3H), 7.40 (br d, 1H),7.36 (m, 2H), 7.28 (s, 1H), 7.22 (d, 1H), 7.06 (s, 2H), 6.95 (d, 1H),5.00 (br d, 2H), 4.95 (s, 2H), 4.70 (t, 1H), 4.39 (m 1H), 4.28 (m, 1H),4.00 (dd, 2R), 3.88 (br in. 2H), 3.85 (br m, 1H), 3.80 (br m, 2H), 3.62(t, 2R), 3.50 (s. 44H), 3.48 (d, 4H), 3.43 (br in. 2H), 3.34 (br m, 2H),3.23 (s, 3H), 3.21 (v br m, 2H), 3.14 (t, 2H), 3.10 (v br m, 1H), 3.00(t, 2H), 2.94 (br m, 1H), 2.76 (v br m, 1H), 2.64 (v br m. 3H), 2.34 (brt, 2H), 2.32 (m, 1H), 2.17 (m, 1H), 2.09 (br d, 3H), 2.00 (br in. 1H),1.56 (br m, 1H), 1.39-1.19 (br in. 8H), 1.19-0.92 (br m. 8H), 0.88 (brd. 3H), 0.87 (br m, 1H), 0.82 (br d. 6H), 0.79 (br s, 3H). MS (ESI) m/e1119.2 [(M−2H)/2]⁻.

2.175 Synthesis of(6S)-2,6-anhydro-6-(2-{2-[{([2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.13,7]dec-1-yl}oxy)ethyl][(3S)-3,4-dihydroxybutyl]carbamoyl}oxy)methyl]-5-({N-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-N-(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yl)-b-alanyl-L-valyl-L-alanyl}amino)phenyl}ethyl)-L-gulonicAcid (Synthon ABU)

The title compound was prepared using the procedure in Example 2.147.4,replacing Example 2.141.4 with Example 2.167.1. MS (ESI) me 1033.4(M+2f)².

2.176 Synthesis of(6S)-2,6-anhydro-6-(2-{2-[({[2-({3-[(4-{6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl]-2-carboxypyridin-3-yl}-5-methyl-1H-pyrazol-1-yl)methyl]-5,7-dimethyltricyclo[3.3.1.13,7]dec-1-yl}oxy)ethyl][(3S)-3,4-dihydroxybutyl]carbamoyl}oxy)methyl]-5-((N-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-N-[2-(2-sulfoethoxy)ethyl]-b-alanyl-L-valyl-L-alanyl)amino)phenyl}ethyl)-L-gulonicAcid (Synthon ABV)

The title compound was prepared using the procedure in Example 2.160.7,replacing Example 2.154.1 with Example 2.167.1. MS (ESI) m/e 859.4(M+2H)²⁺.

Example 3: Generation of Mouse Anti-B7-H3 Monoclonal Antibodies by MouseHybridoma Technology

B7-H3 specific antibodies were raised using mouse hybridomas technology.Specifically, a mouse fibroblast cell line (3T12) expressing full lengthhuman B7-H3 as well as recombinant human or mouse B7-H3-ECD-human Fcfusion proteins were used as immunogens, the sequences of which areprovided in Table 1 Human HCT16 cell lines expressing human B7-H3 wereused for determining anti-sera titer and for screening antigen-specificantibodies. Cell lines were exposed to approximately 3000 mREM of gammasource radiation prior to immunization. Two different strains of micewere immunized in the hock with dosages containing 5×10⁶cells/mouse/injection or 10 ug of protein/mouse/inject ion in thepresence of Gerbu MM adjuvant (Cooper-Casey Corporation, Valley Center,CA, US) for both primary and boost immunizations. To increase immuneresponse to mouse B7-H3, the mice were further boosted with a mixture ofhuman and mouse B7-H3-ECD-human Fc proteins for the final boosts.Briefly, the antigens were prepared in PBS as follows: 200×10⁶ cells/mLor 400 ug/mL protein. The calculated volume of antigen was transferredto a sterile microcentrifuge tube and equal volume of Gerbu MM was thenadded. The solution was mixed by gently vortexing for 1 minute. Theadjuvant-antigen solution was then drawn into a proper syringe foranimal injection. A total of 25 μL of the mixture was injected into thehock of each leg of the mouse. Each animal was boosted 3 times beforeserum titer was determined for the groups. All animals were given 2additional boosts with an equal mixture of mouse B7-H3-ECD-human Fc andhuman B7-H3-ECD-human Fc proteins in adjuvant before fusion.

TABLE 1 Amino acid sequences of recombinantproteins used for immunization or screening Protein Amino Acid SequenceHuman full MLRRRGSPGMGVHVGAALGALWFCLTGALEVQVPEDPVVALVGTDATLCCSFSPEPGlength B7-H3 FSLAQLNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYQGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSILRVVLGANGTYSCLVRNPVLQQDAHSSVTITPQRSPTGAVEVQVPEDPVVALVGTDATLRCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYRGYPEAEVFWQDGQGVPLIGNVITSQMANEQGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQPMTFPPEALWVTVGLSVCLIALLVALAFVCWRKIKQSCEEENAGAEDQDGEGEGSKTALQPLKHSDSKEDDGQEIA (SEQ ID NO: 149) Human B7-H3-MLRRRGSPGMGVHVGAALGALWFCLTGALEVQVPEDPVVALVGTDATLCCSFSPEPG ECD (fcFSLAQLNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQRVRV fusion)ADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYQGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSILRVVLGANGTYSCLVRNPVLQQDAHSSVTITPQRSPTGAVEVQVPEDPVVALVGTDATLRCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYRGYPEAEVFWQDGQGVPLIGNVITSQMANEQGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQPMTFAAADKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVICVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 150) Mouse B7-H3-MLRGWGGPSVGVCVRTALGVLCLCLTGAVEVQVSEDPVVALVDTDATLRCSFSPEPG ECD (fcFSLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYSNRTALFPDLLVQGNASLRLQRVRV fusion)TDEGSYTCFVSIQDFDSAAVSLQVAAPYSKPSMTLEPNKDLRPGNMVTITCSSYQGYPEAEVFWKDGQGVPLTGNVTTSQMANERGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQPLTFAAADKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVICVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 151) Human B7-H3-MEFGLSWLFLVAILKGVQCGALEVQVPEDPVVALVGTDATLCCSFSPEPGFSLAQLN ECD (HisLIWQLTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFT tag)CFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYQGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSILRVVLGANGTYSCLVRNPVLQQDAHSSVTITPQRSPTGAVEVQVPEDPVVALVGTDATLRCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYRGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQPMTHHHHHH (SEQ ID NO: 152) Mouse B7-H3-MEFGLSWLFLVAILKGVQCVEVQVSEDPVVALVDTDATLRCSFSPEPGFSLAQLNLI ECD (HisWQLTDTKQLVHSFTEGRDQGSAYSNRTALFPDLLVQGNASLRLQRVRVTDEGSYTCF tag)VSIQDFDSAAVSLQVAAPYSKPSMTLEPNKDLRPGNMVTITCSSYQGYPEAEVFWKDGQGVPLIGNVITSQMANERGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQPLTFHHHHHH (SEQ ID NO: 153) Cyno B7-H3-MLHRRGSPGMGVHVGAALGALWFCLTGALEVQVPEDPVVALVGTDATLRCSFSPEPG ECD (hisFSLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYANRTALFLDLLAQGNASLRLQRVRV tag)ADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYRGYPEAEVFWQDGQGAPLTGNVTTSQMANEQGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDAHGSITITPQRSPTGAVEVQVPEDPVVALVGTDATLRCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYANRTALFLDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYRGYPEAEVFWQDGQGAPLIGNVITSQMANEQGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQPMTFAAAHHHHHHHH (SEQ ID NO: 154) Note: leader sequence, Fc, and Hissequences are underlined

Hybridoma Fusion and Screening

Cells of murine myeloma cell line (NS-0, ECACC No. 85110503) werecultured to reach the log phase stage right before fusion. Popliteal andinguinal lymph nodes were removed from each mouse and single cellsuspensions were prepared sterilely. Lymphocytes were fused with myelomacells (E. Harlow, D. Lane, Antibody: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1998): Kohler G, andMilstein C., “Continuous cultures of fused cell secreting antibody ofpredefined specificity,” Nature, 256:495497 (1975); BTX HarvardApparatus (Holliston, Mass., US) ECM 2001 technical manual). Fusedhybrid cells were dispensed into 96-well plates in DMEM/10% FBS/HATmedia. Supernatants from surviving hybridoma colonies were subjected tocell-based screening using human cell lines expressing the recombinanthuman B7-H3. Briefly, a human cell line expressing the human B7-H3 wasthawed and directly dispensed into 96 well (black with clear bottom forimaging) plates at 50,000 cells/well in growth media and incubated for 2days at 37° C. to reach 50% confluency. Hybridoma supernatants (50μL/well) were transferred to respective plates and incubated at roomtemperature for 30 minutes. Media was removed from each well and goatanti-mouse IgG-AF488 (Invitrogen, No. A11029, Grand Island, N.Y. US) wasused for detection using the InCell Analyzer 2000 (GE). Hits wereexpanded and binding was confirmed by FACS using a different human cellline or a mouse cell line expressing the human B7-H3 and goat anti-mouseIgG-PE for detection. Species specificity was determined using the ELISAformat according to the following procedure. ELISA plates were coatedwith human B7-H3-ECD-human Fc, cynomolgous B7-H3-ECD-his, or mouseB7-H3-ECD-human Fc proteins overnight at room temperature. Plates werewashed and hybridoma supes (100 μL) was added to each well, andincubated at room temperature for 1 hour. Plates were washed, donkeyanti-mouse IgG-HRP (Jackson Immunochemicals, No. 115-035-071. WestGrove, Pa., US) was used for detection, and binding ODs were observed at650 nm.

A selection of hits were subcloned using the MoFlo (Beckman.Indianapolis, Ind. US) by depositing a single cell per well into % wellcell culture plates to ensure clonality of the cell line. Resultingcolonies were screened for specificity by FACS using mouse 3T12fibroblast cell lines expressing the human B7-H3, cynomolgous B7-H3 ormouse B7-H3. Isotype of each monoclonal antibody was determined usingthe Mouse Monoclonal Isostyping Kit (Roche, No. 11-493-027-001,Indianapolis. Ind., USA). Hybridoma clones producing antibodies thatshowed high specific binding activity against human and cynomolgus B7-H3antigen were subcloned and purified (Table 2).

TABLE 2 List of Anti-B7-H3 antibodies generated using mouse hybridomatechnology FACS Binding (EC₅₀ nM) Clone Cynomolgous Name Species/IsotypeHuman B7-H3 B7-H3 Mouse B7-H3 Ab1 mouse IgG1/k 2.10 1.79 299.0 Ab2 mouseIgG1/k 1.70 1.50 1.00 Ab3 mouse IgG1/k 1.66 1.42 0.94 Ab4 mouse IgG2b/k4.06 3.10 1.75 Ab5 mouse IgG1/k 2.71 1.91 6.01 Ab6 mouse IgG1/k 1.591.53 No binding Ab7 mouse IgG1/k 3.22 2.67 67.13 Ab8 mouse IgG1/k 3.838.63 193.0 Ab9 mouse IgG1/k 4.49 259.0 0.72 Ab10 mouse IgG2b/k 3.97 4.463.80 Ab11 mouse IgG1/k 23.40 2.03 568.60 Ab12 mouse IgG1/k 3.88 6.718.72 Ab13 mouse IgG1/k 1.94 4.12 25.80 Ab14 mouse IgG1/k 3.03 2.97 102.2Ab15 mouse IgG1/k 5.37 6.52 4.61 Ab16 mouse IgG1/k 3.94 4.28 318.7 Ab17mouse IgG2b/k 2.75 2.60 2.39 Ab18 mouse IgG1/k 5.98 6.49 No binding

Example 4: In Vitro Characterization of Anti-B7-H3 Mouse MonoclonalAntibodies

The binding affinity of the purified anti-B7-H3 monoclonal antibodieswas determined by surface plasma resonance. Table 3 shows theassociation rate constants (k_(a)) dissociation rate constants (k_(d))and equilibrium dissociation constants (K_(D)) for a series of mousehybridoma derived anti-B7-H3 monoclonal antibodies (mAbs) binding to thesoluble ECDs of human B7-H3 and cyno B7-H3. The binding kinetics werederived from SPR measurements using a Biacore T200 instrument and a mAbcapture approach (as described in the materials and methods below).

TABLE 3 Biacore kinetics of anti-B7-H3 mouse hybridoma antibodiesbinding to human and cynomolgus monkey B7-H3. Murine Antibody huB7-H3cynoB7-H3 Name k_(a) (1/Ms) k_(d) (1/s) K_(D) (M) k_(a) (1/Ms) k_(d)(1/s) K_(D) (M) Ab17 5.4E+05 1.9E−05 3.4E−11 5.1E+05 1.0E−05 1.9E−11Ab18 2.1E+05 3.6E−05 1.7E−10 2.4E+05 2.9E−05 1.2E−10 Ab15 8.0E+043.4E−05 4.3E−10 7.7E+04 7.0E−05 9.1E−10 Ab4 6.9E+05 1.1E−03 1.6E−095.4E+05 9.6E−04 1.8E−09 Ab8 5.8E+04 9.9E−05 1.7E−09 1.6E+05 2.6E−041.7E−09 Ab10 4.1E+04 1.9E−04 4.6E−09 2.0E+05 4.2E−03 2.0E−08 Ab123.8E+04 2.5E−04 6.7E−09 5.5E+04 1.0E−05 1.8E−10 Ab5 1.3E+06 1.2E−029.2E−09 1.4E+06 2.8E−01 2.0E−07 Ab14 1.1E+05 1.4E−03 1.3E−08 6.9E+053.0E−03 4.3E−09 Ab9 6.6E+04 1.1E−03 1.7E−08 poor kinetic fit Ab133.3E+05 5.8E−03 1.7E−08 4.4E+05 3.7E−03 8.4E−09 Ab3 5.2E+05 1.0E−021.9E−08 3.8E+05 1.0E−02 2.6E−08 Ab16 1.4E+05 3.2E−03 2.4E−08 7.5E+055.6E−03 7.5E−09 Ab2 1.2E+05 2.9E−03 2.4E−08 2.3E+05 1.1E−02 5.0E−08 Ab112.0E+04 8.9E−04 4.5E−08 2.7E+04 7.2E−05 2.6E−09 Ab6 1.2E+04 1.0E−028.4E−07 2.8E+04 1.2E−02 4.1E−07 Ab1 no no observable observable bindingbinding Ab7 little little observable observable binding binding

Pair-wise binding assays performed on Biacore T200 SPR instruments wereused to determine the relative epitope grouping for the murineanti-B7-H3 mAbs as described in the methods below. FIG. 1 shows anepitope grouping depiction, which describes the relative human B7-H3epitope diversity and overlap for a series of anti-B7-H3 mAbs identifiedherein. Epitope groups are represented as individual ovals, some ofwhich overlap with each other. Antibodies in different epitope groupscan bind to B7-H3 simultaneously and likely bind to different epitopeswhile antibodies within a given epitope group cannot bind to B7-H3simultaneously and likely bind to overlapping epitopes. The groupinginformation was derived from a simultaneous binding assay as describedin materials and methods. Ab3, Ab4, Ab5, Ab11, Ab12, and Ab8 groupingswere ambiguous.

Materials and Methods: Binding Kinetics

Biacore T200 SPR instruments were used to measure the binding kineticsof human B7-H3 (analyte) binding to various mAbs (ligands). The assayformat was Fc-based capture via immobilized anti-mouse (Fc) (Pierce31170) or immobilized anti-human (Fc) (Pierce 31125). A standard aminecoupling protocol was employed to immobilize the capture reagents viaprimary amines to the carboxy-methyl (CM) dextran surface of CM5sensorchips (Biacore); capture antibodies were coupled to a level ofapproximately 5000RU. For binding kinetic measurements the assay bufferwas HBS-EP+ (Biacore): 10 mM Hepes, pH7.4, 150 mM NaCl, 3 mM EDTA. 0.05%polysorbate 20. During the assay, all measurements were referencedagainst the capture surface alone. Each assay cycle consisted of thefollowing steps: 1) Capture of ligand to approximately 50RU; 2) Analyteinjection over both reference and test surface. 240 μL at 80 μL/min,after which the dissociation was monitored for 900 seconds at 80 μL/min;3) Regeneration of capture surface with low pH glycine. For kineticdeterminations analyte injections were 3-point. 9-fold dilution seriesof 900 nM, 100 nM and 11.11 nM, buffer only injections were included forsecondary referencing. Data were processed and fit to a 1:1 bindingmodel using Biacore T200 Evaluation Software to determine the bindingkinetic rate constants, k_(a) (on-rate) and k_(d) (off-rate), and theequilibrium dissociation constant (affinity, K_(D)).

Materials and Methods: Epitope Grouping

Pair-wise binding assays performed on Biacore T200 SPR instruments wereused to determine the relative epitope grouping for a series ofanti-B7-H3 mAbs. The assay format was Fc-based capture via immobilizedanti-mouse (Fc) (Pierce 31170) or immobilized anti-human (Fc) (Pierce31125). A standard amine coupling protocol was employed to immobilizethe capture reagents via primary amines to the carboxy-methyl (CM)dextran surface of CM5 sensorchips (Biacore); capture antibodies werecoupled to a level of approximately 2000RU. Epitope groupingmeasurements were done at 12° C. (low temperature allows for groupinginformation on fast off-rate mAbs), the assay buffer wasHBS-EP+(Biacore): 10 mM Hepes, pH7.4, 150 mM NaCl, 3 mM EDTA. 0.05%polysorbate 20. Each assay cycle consisted of the following steps in afour flowcell system: 1) separate test mAbs were captured in flowcells2, 3 & 4 (flowcell 1 was reference, no test mAb); 2) all 4 flowcellswere then blocked by injection with isotype control mAb or isotype mAbcocktail at 50 μg/mL; 3) all 4 flowcells were then injected with antigenor buffer only (buffer only is for double referencing, done for each mAbpair individually); 4) all 4 flowcells were then injected with 2nd testmAb at 10 μg/mL; 5) all 4 flowcells were then regenerated with glycine,pH 1.5. The assay was done for each test mAb pair in reciprocalorientations. Simultaneous binding was evaluated examining the ratio ofthe 2nd test mAb response to the Ag response (RU_(mAb2)/RU_(Ag)); ifthis ratio was equal to or greater than 0.2 the interaction was scoredas a simultaneous binder. From this pair-wise binding assay data a“venn” style diagram was constructed manually to depict relative epitopegroupings.

Example 5: Generation of Anti-hB7-H3 Chimeric Antibodies

Following the identification of mouse anti-B7-H3 hybridoma antibodies,heavy and light chain variable regions (VH and VL) corresponding to thesecreted antibodies were determined from cells using reversetranscriptase-polymerase chain reaction (RT-PCR). Murine variableregions were expressed in mammalian host cells in the context of a humanimmunoglobulin constant region to provide chimeric antibodies. Table 4below provides the variable region amino acid sequences for the mousechimerized hybridomas.

TABLE 4 Variable region amino acid sequences ofanti-B7-H3 antibodies from mouse hybridomas SEQ Protein ID NO: CloneRegion Residues Amino Acid Sequence 1 chAb2 VHQVQLQQPGAELVKPGASVKLSCKASGY TFTSYWMHWVKQRPGQGLEWIGMIHPDSGTTNYNEKFRSKATLTVDKSSSTAYM QLSSLTSEDSAVYYCAVYYGSTYWYFD VWGTGTTVTVSS 2chAb2 CDR-H1 Residues 26-35 GYTFTSYWMH of SEQ ID NO: 1 3 chAb2 CDR-H2Residues 50-66 MIHPDSGTTNYNEKFRS of SEQ ID NO: 1 4 chAb2 CDR-H3Residues 99-109 YYGSTYWYFDV of SEQ ID NO: 1 5 chAb2 VLDVVMTQTPLSLPVSLGDQAYISCRSSQ SLVHINGNTYLHWYRQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKIS RVEAEDLGVYFCSQSTHFPFTFGSGTK LEIK 6 chAb2CDR-L1 Residues 24-39 RSSQSLVHINGNTYLH of SEQ ID NO: 5 7 chAb2 CDR-L2Residues 55-61 KVSNRFS of SEQ ID NO: 5 8 chAb2 CDR-L3 Residues 94-102SQSTHFPFT of SEQ ID NO: 5 9 chAb3 VH QVQLQQPGAELVKPGASVKLSCKASGYTFSSYWMHWVKQRPGQGLEWIGLIHPD SGSTNYNEMFKNKATLTVDRSSSTAYVQLSSLTSEDSAVYFCAGGGRLYFDYWG QGTTLTVSS 10 chAb3 CDR-H1 Residues 26-35GYTFSSYWMH of SEQ ID NO: 9 11 chAb3 CDR-H2 Residues 50-66LIHPDSGSTNYNEMFKN of SEQ ID NO: 9 12 chAb3 CDR-H3 Residues 99-106GGRLYFDY of SEQ ID NO: 9 13 chAb3 VL DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGDTYLRWYLQKPGQSPKLLIY KVSNRFSGVPDRFSGSGSGTDFTLKITRVEAEDLGVYFCSQSTHVPYTFGGGTK LEIK 14 chAb3 CDR-L1 Residues 24-39RSSQSLVHSNGDTYLR of SEQ ID NO: 13 7 chAb3 CDR-L2 Residues 55-61 KVSNRFSof SEQ ID NO: 13 15 chAb3 CDR-L3 Residues 94-102 SQSTHVPYTof SEQ ID NO: 13 16 chAb4 VH QVQLQQPGAELVKPGASVKLSCKASGYSFTSYWMHWVKQRPGQGLEWIGMIHPN SGSNNYNEKFKSKATLTVDKSSNTAYMQLSSLTSEDSAVYYCARRLGLHFDYWG QGTTLTVSS 17 chAb4 CDR-H1 Residues 26-35GYSFTSYWMH of SEQ ID NO: 16 18 chAb4 CDR-H2 Residues 50-66MIHPNSGSNNYNEKFKS of SEQ ID NO: 16 19 chAb4 CDR-H3 Residues 99-106RLGLHFDY of SEQ ID NO: 16 20 chAb4 VL DIVMTQSQKFMSTPVGDRVSITCKASQNVGTAVAWYQQKPGQSPKLLIYSASNR YTGVPDRFTGSGSGTDFTLTISNMQSEDLADYFCQQYSSYPYTFGGGTKLEIK 21 chAb4 CDR-L1 Residues 24-34 KASQNVGTAVAof SEQ ID NO: 20 22 chAb4 CDR-L2 Residues 50-56 SASNRYT of SEQ ID NO: 2023 chAb4 CDR-L3 Residues 89-97 QQYSSYPYT of SEQ ID NO: 20 24 chAb18 VHQVQLQQSAAELARPGASVKMSCKASGY SFTSYTIHWVKQRPGQGLEWIGYINPNSRNTDYNQKFKDETTLTADRSSSTAYM QLISLTSEDSAVYYCARYSGSTPYWYF DVWGAGTTVTVSS 25chAb18 CDR-H1 Residues 26-35 GYSFTSYTIH of SEQ ID NO: 24 26 chAb18CDR-H2 Residues 50-66 YINPNSRNTDYNQKFKD of SEQ ID NO: 24 27 chAb18CDR-H3 Residues 99-110 YSGSTPYWYFDV of SEQ ID NO: 24 28 chAb18 VLQIVLTQSPAILSASPGEKVTMTCRASS SVSYMNWYQQKPGSSPKPWIYATSNLASGVPARFSVSVSGTSHSLTISRVEAED AATYYCQQWSSNPLTFGAGTKLELK 29 chAb18 CDR-L1Residues 24-33 RASSSVSYMN of SEQ ID NO: 28 30 chAb18 CDR-L2Residues 49-55 ATSNLAS of SEQ ID NO: 28 31 chAb18 CDR-L3 Residues 88-96QQWSSNPLT of SEQ ID NO: 28 32 chAb13 VH DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYSWHWIRQFPGNKLEWMGYIHS SGSTNYNPSLKSRISINRDTSKNQFFLQLNSVTTEDTATYYCAGYDDYFEYWGQ GTTLTVSS 33 chAb13 CDR-H1 Residues 26-36GYSITSGYSWH of SEQ ID NO: 32 34 chAb13 CDR-H2 Residues 51-66YIHSSGSTNYNPSLKS of SEQ ID NO: 32 35 chAb13 CDR-H3 Residues 99-105YDDYFEY of SEQ ID NO: 32 36 chAb13 VL DIVMTQSQKFMSTSVGDRVSVTCKASQNVGFNVAWYQQKPGQSPKALIYSASYR YSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYPFTFGSGTKLEIK 37 chAb13 CDR-L1 Residues 24-34 KASQNVGFNVAof SEQ ID NO: 36 38 chAb13 CDR-L2 Residues 50-56 SASYRYSof SEQ ID NO: 36 182 enAb13 CDR-L3 Residues 89-97 QQYNSYPFTof SEQ ID NO: 36 40 chAb12 VH EVQLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEKRLEWVATISSG TNYTYYPDSVKGRFTISRDNAKNTLYLQMTSLRSEDTAMYYCARQGRYSWIAYW GQGTLVTVSA 41 chAb12 CDR-H1 Residues 26-35GFTFSSYAMS of SEQ ID NO: 40 42 chAb12 CDR-H2 Residues 50-66TISSGTNYTYYPDSVKG of SEQ ID NO: 40 43 chAb12 CDR-H3 Residues 99-107QGRYSWIAY of SEQ ID NO: 40 44 chAb12 VL DIVLTQSPASLAVSLGQRATISCRASKSVSTSDYSYMHWNQQKPGQPPKLLIYL ASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELLTFGAGTKLE LK 45 chAb12 CDR-L1 Residues 24-38RASKSVSTSDYSYMH of SEQ ID NO: 44 46 chAb12 CDR-L2 Residues 54-60 LASNLESof SEQ ID NO: 44 47 chAb12 CDR-L3 Residues 93-100 QHSRELLTof SEQ ID NO: 44 48 chAb14 VH EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPEKRLEWVATISGG GTNTYYPDSVEGRFTISRDNAKNFLYLQMSSLRSEDTALYYCARHYGSQTMDYW GQGTSVTVSS 49 chAb14 CDR-H1 Residues 26-35GFTFSSYGMS of SEQ ID NO: 48 50 chAb14 CDR-H2 Residues 50-66TISGGGTNTYYPDSVEG of SEQ ID NO: 48 51 chAb14 CDR-H3 Residues 99-107HYGSQTMDY of SEQ ID NO: 48 52 chAb14 VL DIQMTQSPASLSASVGETVTITCRTSGNIHNYLTWYQQKQGKSPQLLVYNAKTL ADGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQHFWSIMWTFGGGTKLEIK 53 chAb14 CDR-L1 Residues 24-34 RTSGNIHNYLTof SEQ ID NO: 52 54 chAb14 CDR-L2 Residues 50-56 NAKTLADof SEQ ID NO: 52 55 chAb14 CDR-L3 Residues 89-97 QHFWSIMWTof SEQ ID NO: 52 56 chAb6 VH QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPG SGSTNYNEKFKGKATFTADTSSNTAYMQVSSLTSEDSAVHYCARRGYGYVPYAL DYWGQGTSVTVSS 57 chAb6 CDR-H1 Residues 26-35GYTFSRYWIE of SEQ ID NO: 56 58 chAb6 CDR-H2 Residues 50-66EILPGSGSTNYNEKFKG of SEQ ID NO: 56 59 chAb6 CDR-H3 Residues 99-110RGYGYVPYALDY of SEQ ID NO: 56 60 chAb6 VL EIQMTQTTSSLSASLGDRVTISCRASQDISNSLNWYQQKPDGTVNLLIYYTSRL YSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIK 61 chAb6 CDR-L1 Residues 24-34 RASQDISNSLNof SEQ ID NO: 60 62 chAb6 CDR-L2 Residues 50-56 YTSRLYS of SEQ ID NO: 6063 chAb6 CDR-L3 Residues 89-97 QQGNTLPYT of SEQ ID NO: 60 64 chAb11 VHEVKLVESGGGLVQPGGSLRLSCATSGF TFTNYYMSWVRQPPGKALEWLGFIRNKANDYTTEYSASVKGRFTISRDNSQSIL YLQMNTLRAEDSATYYCARESPGNPFA YWGQGTLVTVSA 65chAb11 CDR-H1 Residues 26-35 GFTFTNYYMS of SEQ ID NO: 64 66 chAb11CDR-H2 Residues 50-68 FIRNKANDYTTEYSASVKG of SEQ ID NO: 64 67 chAb11CDR-H3 Residues 101-109 ESPGNPFAY of SEQ ID NO: 64 68 chAb11 VLDIVMTQSPSSLTVTAGEKVTMTCKSSQ SLLNSGTQKNFLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTI SSVQAEDLAVYFCQNDYIYPLTFGAGT KLELK 69 chAb11CDR-L1 Residues 24-40 KSSQSLLNSGTQKNFLT of SEQ ID NO: 68 70 chAb11CDR-L2 Residues 56-62 WASTRES of SEQ ID NO: 68 71 chAb11 CDR-L3Residues 95-103 QNDYIYPLT of SEQ ID NO: 68 72 chAb16 VHEVKLLESGGGLVQPGGSLKLSCAASGF DFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYTPSLKDKFIISRDNAKNTLYL QMSKVRSEDTALYYCARPGFGNYIYAM DYWGQGTSVTVSS 73chAb16 CDR-H1 Residues 26-35 GFDFSRYWMS of SEQ ID NO: 72 74 chAb16CDR-H2 Residues 50-66 EINPDSSTINYTPSLKD of SEQ ID NO: 72 75 chAb16CDR-H3 Residues 99-110 PGFGNYIYAMDY of SEQ ID NO: 72 76 chAb16 VLDIQMTQTTSSLSASLGDRVTINCRASQ DISNFLNWYQQKPDGTVKLLIYYTSRLYLGVPSRFSGSGSGTDYSLTISNLEQE DIATYFCQQGNTLPPTFGGGTKLEIK 77 chAb16 CDR-L1Residues 24-34 RASQDISNFLN of SEQ ID NO: 76 78 chAb16 CDR-L2Residues 50-56 YTSRLYL of SEQ ID NO: 76 79 chAb16 CDR-L3 Residues 89-97QQGNTLPPT of SEQ ID NO: 76 80 chAb10 VH DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNRLEWMGHINY SGITNYNPSLKSRISITRDTSKNQFFLQLYSVTTEDTATYFCARRSLFYYYGSS LYAMDYWGQGTSVIVSS 81 chAb10 CDR-H1Residues 26-36 GYSITSDYAWN of SEQ ID NO: 80 82 chAb10 CDR-H2Residues 51-66 HINYSGITNYNPSLKS of SEQ ID NO: 80 83 chAb10 CDR-H3Residues 99-114 RSLFYYYGSSLYAMDY of SEQ ID NO: 80 84 chAb10 VLDVVMTQSPFSLPVSLGDQASISCRSSQ SLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKIS RVEAEDLGVYFCSQSTHVPWTFGGGTK LEIK 85 chAb10CDR-L1 Residues 24-39 RSSQSLVHSNGNTYLH of SEQ ID NO: 84 7 chAb10 CDR-L2Residues 55-61 KVSNRFS of SEQ ID NO: 84 86 chAb10 CDR-L3 Residues 94-102SQSTHVPWT of SEQ ID NO: 84 87 chAb7 VH EVQLVESGENLVKPGGSLKLSCAASGFSFRGYGMSWVRQTPDKRLEWVAAISTG GNYTYYPDSVQGRFTISRDNANNTLYLQMSSLKSEDTAMYYCARRGGNYAGFAY WGQGTLVTVSA 88 chAb7 CDR-H1 Residues 26-35GFSFRGYGMS of SEQ ID NO: 87 89 chAb7 CDR-H2 Residues 50-66AISTGGNYTYYPDSVQG of SEQ ID NO: 87 90 chAb7 CDR-H3 Residues 99-108RGGNYAGFAY of SEQ ID NO: 87 91 chAb7 VL DIQMTQSPASLSVSVGETVTITCRPSENIYSNLAWYQQKQGKSPQLLVYAATNL ADGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHFWGTPFTFGSGTKLEIK 92 chAb7 CDR-L1 Residues 24-34 RPSENIYSNLAof SEQ ID NO: 91 93 chAb7 CDR-L2 Residues 50-56 AATNLAD of SEQ ID NO: 9194 chAb7 CDR-L3 Residues 89-97 QHFWGTPFT of SEQ ID NO: 91 95 chAb8 VHEVKLVESGGGLVKPGGSLKLSCAASGF TFSSYGMSWVRQTPEKRLEWVATISGGGNYTYCPDSVKGRFTISRDNAKNNLYL QMSSLRSEDTALYYCTRQRGYDYHYAM DFWGQGTSVTVSS 49chAb8 CDR-H1 Residues 26-35 GFTFSSYGMS of SEQ ID NO: 95 96 chAb8 CDR-H2Residues 50-66 TISGGGNYTYCPDSVKG of SEQ ID NO: 95 97 chAb8 CDR-H3Residues 99-110 QRGYDYHYAMDF of SEQ ID NO: 95 98 chAb8 VLDIQMTQSPASLSVSVGETVTITCRASE NIYSNLAWHQQKQGKSPQLLVYAATNLADGVPSRFSGNGSDTQYSLKINSLQSE DFGSYFCQNFWGTSWTFGGGTKLEIK 99 chAb8 CDR-L1Residues 24-34 RASENIYSNLA of SEQ ID NO: 98 93 chAb8 CDR-L2Residues 50-56 AATNLAD of SEQ ID NO: 98 100 chAb8 CDR-L3 Residues 89-97QNFWGTSWT of SEQ ID NO: 98 101 chAb17 VH EVKLVESGGGLVQPGGSLKLSCAASGFTFSSYIMSWVRQTPEKRLEWVASIVSS NITYYPDSMKGRFTISRDNARNILYLQMSSLKSEDTAMYYCARSGTRAWFAYWG QGTLVTVSA 102 chAb17 CDR-H1 Residues 26-35GFTFSSYIMS of SEQ ID NO: 101 103 chAb17 CDR-H2 Residues 50-65SIVSSNITYYPDSMKG of SEQ ID NO: 101 104 chAb17 CDR-H3 Residues 98-106SGTRAWFAY of SEQ ID NO: 101 105 chAb17 VL DIVLTQSPASLAVSLGQRATISCRASKSVSTSAYSYMHWYQQKPGQPPKLLIYL ASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPYTFGGGTKL EIK 106 chAb17 CDR-L1 Residues 24-38RASKSVSTSAYSYMH of SEQ ID NO: 105 46 chAb17 CDR-L2 Residues 54-60LASNLES of SEQ ID NO: 105 107 chAb17 CDR-L3 Residues 93-101 QHSRELPYTof SEQ ID NO: 105 108 chAb5 VH QVQLQQPGDELVKPGASVKLSCKTSGYTFTTDWMHWVKQRPGQGLEWIGMIHPN SGTTNYNEKFKSKAALTVDKSSSTACMQLSSLTSEDSAVYYCARSYWKWYFDVW GTGTTVTVSS 109 chAb5 CDR-H1 Residues 26-35GYTFTTDWMH of SEQ ID NO: 108 110 chAb5 CDR-H2 Residues 50-66MIHPNSGTTNYNEKFKS of SEQ ID NO: 108 111 chAb5 CDR-H3 Residues 99-107SYWKWYFDV of SEQ ID NO: 108 112 chAb5 VL QIVLTQSPAIMSASLGEEITLTCSASSSVSYMHWYQQKSGTSPKLLIYSTSNLA SGVPSRFSGSGSGTFYSLTISSVEAEDSADYYCHQWTSYMYTFGGGTKLEIK 113 chAb5 CDR-L1 Residues 24-33 SASSSVSYMHof SEQ ID NO: 112 114 chAb5 CDR-L2 Residues 49-55 STSNLASof SEQ ID NO: 112 115 chAb5 CDR-L3 Residues 88-96 HQWTSYMYTof SEQ ID NO: 112

Example 6: Binding Characterization of Chimeric Anti-B7-H3 Antibodies

To generate purified chimeric antibodies, expression vectors weretransiently transfected into HEK293 6E suspension cell cultures in aratio of 60% to 40% light to heavy chain construct. 1 mg/ml ofpolyethylenimine (PEI) or 2.6 μL/mL of Expifectamine was used totransfect the cells. Cell supernatants were harvested after five days inshaking flasks, spun down to pellet cells, and filtered through 0.22 μmfilters to separate IgG from culture contaminants. Antibody-containingsupernatants were purified on Akta Pure using protein A mAb SelectSure.Columns were equilibrated in PBS pH 7.4, supernatants were then passedthrough the column and a wash was performed with PBS pH 7.4.

IgG were eluted with 0.1 M acetic acid pH 3.5 and collected in severalaliquots. Fractions containing IgG were pooled and dialyzed in PBSovernight at 4° C. Anti-B7-H3 chimeric antibodies that were successfullyexpressed were characterized for the ability to bind the B7-H3overexpressing human non-small cell lung cancer cell line NCI-H1650(ATCC® No. CRL-5883) by FACS using the methods described below. Table 5summarizes the binding properties of the chimeric anti-B7-H3 antibodies.

TABLE 5 In vitro characterization of B7-H3 chimeric antibodies ParentalFACS binding Chimeric Ab Name Isotype Hybridoma (EC₅₀ nM) chAb2 huIgG1/kAb2 0.10 chAb3 huIgG1/k Ab3 0.61 chAb4 huIgG1/k Ab4 0.56 chAb18 huIgG1/kAb18 1.14 chAb13 huIgG1/k Ab13 1.53 chAb11 huIgG1/k Ab11 1.12 chAb6huIgG1/k Ab6 0.33 chAb16 huIgG1/k Ab16 0.27 chAb14 huIgG1/k Ab14 0.81

FACS Binding Methods

Cells were harvested from flasks when approximately 80% confluent usingGibco® Cell Dissociation Buffer. Cells were washed once in PBS/l % FBS(FACS buffer) then resuspended at 2.5×10⁶ cells/mL in FACS buffer. 100μL of cells/well were added to around bottom 96-well plate. 10 μL of a10× concentration of mAb/ADC (final concentrations are indicated thefigures). Wells were washed twice with FACS buffer and resuspended in 50μL of secondary Ab (AlexaFluor 488) diluted in FACS buffer. The platewas incubated at 4° C. for one hour and washed twice with FACS buffer.Cells were resuspended in 100 μL of PBS/1% formaldehyde and analyzed ona Becton Dickinson LSRII flow cytometer. Data was analyzed using WinListflow cytometry analysis software.

Example 7: Characterization of Chimeric Anti-B7-H3 Antibodies as Bcl-xLInhibiting Antibody Drug Conjugates

Nine anti-B7-H3 chimeric antibodies were conjugated to the Bcl-xLinhibiting (Bcl-xLi) synthon CZ (Example 2.1) using conjugation Method Adescribed below. The resulting ADCs (anti-B7-H3 antibodies conjugated tosynthon CZ) were tested for binding to cell surface human B7-H3 by FACS(as described in Example 6) and for cell cytotoxicity in cell linesexpressing B7-H3. Of the nine antibodies, three antibodies (chAb2,chAb6, and chAb16) precipitated following conjugation to synthon CZ andshowed weak cytotoxicity in cells expressing human B7-H3. Table 6provides cell surface binding and cytotoxicity activity of anti-B7-H3chimera ADCs against breast cancer cell HCC38 expressing human B7-H3.

TABLE 6 In vitro characterization of B7-H3 chimeric-CZ conjugates % FACSBinding Cytotoxicity DAR agg Conjugation Human B7-H3 (HCC38 cellConjugation by by ADC Name observation EC₅₀ nM line IC₅₀ nM) Method MSSEC chAb2-CZ Precipitates 2.60 4.77 A 1.0 5.9 chAb3-CZ Clear 0.65 0.17 A4.6 6.3 chAb4-CZ Clear 0.54 0.26 A 1.4 5.7 chAb18-CZ Clear 1.78 0.28 A2.3 4.3 chAb13-CZ Clear 1.49 0.18 A 3.8 8.3 chAb11-CZ Clear 1.12 2.34 A3.2 5.6 chAb6-CZ Precipitates 0.56 80.98 A 1.3 0.5 chAb16-CZPrecipitates 0.62 21.89 A 0.9 2.3 chAb14-CZ Clear 0.50 2.01 A 1.4 1.9

Materials and Methods: Conjugation of Bcl-xL Inhibitory ADCs

ADCs were synthesized using one of the methods described below.Exemplary ADCs were synthesized using one of nine exemplary methods,described below.

Method A.

A solution of Bond-Breaker™ tris(2-carboxyethyl)phosphine (TCEP)solution (10 mM, 0.017 mL) was added to a solution of antibody (10mg/mL, 1 mL) preheated to 37° C. The reaction mixture was kept at 37° C.for 1 hour. The solution of reduced antibody was added to a solution ofsynthon (3.3 mM. 0.160 mL in DMSO) and gently mixed for 30 minutes. Thereaction solution was loaded onto a desalting column (PD10, washed withDulbecco's phosphate-buffered saline [DPBS]3× before use), followed byDPBS (3 mL). The purified ADC solution was filtered through a 0.2micron, low protein-binding 13 mm syringe-filter and stored at 4° C.

Method B.

A solution of Bond-Breaker™ tris(2-carboxyethyl)phosphine (TCEP)solution (10 mM, 0.017 mL) was added to the solution of antibody (10mg/mL, 1 mL) preheated to 37° C. The reaction mixture was kept at 37° C.for 1 hour. The solution of reduced antibody was adjusted to pH=8 byadding boric buffer (0.05 mL, 0.5 M. pH 8), added to a solution ofsynthon (3.3 mM. 0.160 mL in DMSO) and gently mixed for 4 hours. Thereaction solution was loaded onto a desalting column (PD10, washed withDPBS 3× before use), followed by DPBS (1.6 mL) and eluted withadditional DPBS (3 mL). The purified ADC solution was filtered through a0.2 micron, low protein-binding 13 mm syringe-filter and stored at 4° C.

Method C.

Conjugations were performed using a PerkinElmer Janus (part AJL8M01)robotic liquid handling system equipped with an I235/96 tip ModuLarDispense Technology (MDT), disposable head (part 70243540) containing agripper arm (part 7400358), and an 8-tip Varispan pipetting arm (part7002357) on an expanded deck. The PerkinElmer Janus system wascontrolled using the WinPREP version 4.8.3.315 Software.

A Pall Filter plate 5052 was pre-wet with 100 μL 1×DPBS using the MDT.Vacuum was applied to the filter plate for 10 seconds and was followedby a 5 second vent to remove DPBS from filter plate. A 50% slurry ofProtein A resin (GE MabSelect Sure) in DPBS was poured into an 8 wellreservoir equipped with a magnetic ball, and the resin was mixed bypassing a traveling magnet underneath the reservoir plate. The 8 tipVarispan arm, equipped with 1 mL conductive tips, was used to aspiratethe resin (250 μL) and transfer to a 96-well filter plate. A vacuum wasapplied for 2 cycles to remove most of the buffer. Using the MDT, 150 μLof 1×PBS was aspirated and dispensed to the 96-well filter plate holdingthe resin. A vacuum was applied, removing the buffer from the resin. Therinse/vacuum cycle was repeated 3 times. A 2 mL, 96-well collectionplate was mounted on the Janus deck, and the MDT transferred 450 μL of5×DPBS to the collection plate for later use. Reduced antibody (2 mg) asa solution in (200 μL) DPBS was prepared as described above forConditions A and preloaded into a 96 well plate. The solutions ofreduced antibody were transferred to the filter plate wells containingthe resin, and the mixture was mixed with the MDT by repeatedaspiration/dispensation of a 100 μL volume within the well for 45seconds per cycle. The aspiration/dispensation cycle was repeated for atotal of 5 times over the course of 5 minutes. A vacuum was applied tothe filter plate for 2 cycles, thereby removing excess antibody. The MDTtips were rinsed with water for 5 cycles (200 μL, 1 mL total volume).The MDT aspirated and dispensed 150 μL of DPBS to the filter plate wellscontaining resin-bound antibody, and a vacuum was applied for twocycles. The wash and vacuum sequence was repeated two more times. Afterthe last vacuum cycle, 100 μL of 1×DPBS was dispensed to the wellscontaining the resin-bound antibody. The MDT then collected 30 μL eachof 3.3 mM dimethyl sulfoxide solutions of synthons plated in a 96-wellformat and dispensed it to the filter plate containing resin-boundantibody in DPBS. The wells containing the conjugation mixture weremixed with the MDT by repeated aspiration/dispensation of a 100 μLvolume within the well for 45 seconds per cycle. Theaspiration/dispensation sequence was repeated for a total of 5 timesover the course of 5 minutes. A vacuum was applied for 2 cycles toremove excess synthon to waste. The MDT tips were rinsed with water for5 cycles (200 μL, 1 mL total volume). The MDT aspirated and dispensedDPBS (150 μL) to the conjugation mixture, and a vacuum was applied fortwo cycles. The wash and vacuum sequence was repeated two more times.The MDT gripper then moved the filter plate and collar to a holdingstation. The MDT placed the 2 mL collection plate containing 450 μL of10×DPBS inside the vacuum manifold. The MDT reassembled the vacuummanifold by placement of the filter plate and collar. The MDT tips wererinsed with water for 5 cycles (200 μL, 1 mL total volume). The MDTaspirated and dispensed 100 μL of IgG Elution Buffer 3.75 (Pierce) tothe conjugation mixture. After one minute, a vacuum was applied for 2cycles, and the eluent was captured in the receiving plate containing450 μL of 5× DPBS. The aspiration/dispensation sequence was repeated 3additional times to deliver ADC samples with concentrations in the rangeof 1.5-2.5 mg/mL at pH 7.4 in DPBS.

Method D.

Conjugations were performed using a PerkinElmer Janus (part AJL8M01)robotic liquid handling system equipped with an 1235/96 tip ModuLarDispense Technology (MDT), disposable head (part 70243540) containing agripper arm (part 7400358), and an 8-tip Varispan pipetting arm (part7002357) on an expanded deck. The PerkinElmer Janus system wascontrolled using the WinPREP version 4.8.3.315 Software.

A Pall Filter plate 5052 was prewet with 100 μL 1×DPBS using the MDT.Vacuum was applied to the filter plate for 10 seconds and was followedby a 5 second vent to remove DPBS from filter plate. A 50% slurry ofProtein A resin (GE MabSelect Sure) in DPBS was poured into an 8-wellreservoir equipped with a magnetic ball, and the resin was mixed bypassing a traveling magnet underneath the reservoir plate. The 8 tipVarispan arm, equipped with 1 mL conductive tips, was used to aspiratethe resin (250 μL) and transfer to a 96-well filter plate. A vacuum wasapplied to the filter plate for 2 cycles to remove most of the buffer.The MDT aspirated and dispensed 150 μL of DPBS to the filter plate wellscontaining the resin. The wash and vacuum sequence was repeated two moretimes. A 2 mL, 96-well collection plate was mounted on the Janus deck,and the MDT transferred 450 μL of 5×DPBS to the collection plate forlater use. Reduced antibody (2 mg) as a solution in (200 μL) DPBS wasprepared as described above for Conditions A and dispensed into the96-well plate. The MDT then collected 30 μL each of 3.3 mM dimethylsulfoxide solutions of synthons plated in a 96-well format and dispensedit to the plate loaded with reduced antibody in DPBS. The mixture wasmixed with the MDT by twice repeated aspiration/dispensation of a 100 μLvolume within the well. After five minutes, the conjugation reactionmixture (230 μL) was transferred to the 96-well filter plate containingthe resin. The wells containing the conjugation mixture and resin weremixed with the MDT by repeated aspiration/dispensation of a 100 μLvolume within the well for 45 seconds per cycle. Theaspiration/dispensation sequence was repeated for a total of 5 timesover the course of 5 minutes. A vacuum was applied for 2 cycles toremove excess synthon and protein to waste. The MDT tips were rinsedwith water for 5 cycles (200 μL, 1 mL total volume). The MDT aspiratedand dispensed DPBS (150 μL) to the conjugation mixture, and a vacuum wasapplied for two cycles. The wash and vacuum sequence was repeated twomore times. The MDT gripper then moved the filter plate and collar to aholding station. The MDT placed the 2 mL collection plate containing 450μL of 10×DPBS inside the vacuum manifold. The MDT reassembled the vacuummanifold by placement of the filter plate and collar. The MDT tips wererinsed with water for 5 cycles (200 μL, 1 mL total volume). The MDTaspirated and dispensed 100 μL of IgG Elution Buffer 3.75 (P) to theconjugation mixture. After one minute, a vacuum was applied for 2cycles, and the eluent was captured in the receiving plate containing450 μL of 5×DPBS. The aspiration/dispensation sequence was repeated 3additional times to deliver ADC samples with concentrations in the rangeof 1.5-2.5 mg/mL at pH 7.4 in DPBS.

Method E.

A solution of Bond-Breaker™ tris(2-carboxyethyl)phosphine (TCEP)solution (10 mM, 0.017 mL) was added to the solution of antibody (10mg/mL, 1 mL) at room temperature. The reaction mixture was heated to 37°C. for 75 minutes. The solution of reduced antibody cooled to roomtemperature and was added to a solution of synthon (10 mM. 0.040 mL inDMSO) followed by addition of boric buffer (0.1 mL. 1M. pH 8). Thereaction solution was let to stand for 3 days at room temperature,loaded onto a desalting column (PD10, washed with DPBS 3×5 mL beforeuse), followed by DPBS (1.6 mL) and eluted with additional DPBS (3 mL).The purified ADC solution was filtered through a 0.2 micron, lowprotein-binding 13 mm syringe-filter and stored at 4° C.

Method F.

Conjugations were performed using a Tecan Freedom Evo robotic liquidhandling system. The solution of antibody (10 mg/mL) was preheated to37° C., and aliquoted to a heated 96 deep-well plate in amounts of 3 mgper well (0.3 mL) and kept at 37° C. A solution of Bond-Breaker™tris(2-carboxyethyl)phosphine (TCEP) solution (1 mM. 0.051 mL/well) wasadded to antibodies, and the reaction mixture was kept at 37° C. for 75minutes. The solution of reduced antibody was transferred to an unheated96 deep-well plate. Corresponding solutions of synthons (5 mM, 0.024 mLin DMSO) were added to the wells with reduced antibodies and treated for15 minutes. The reaction solutions were loaded onto a platform (8×12) ofdesalting columns (NAP5, washed with DPBS 4× before use), followed byDPBS (0.3 mL) and eluted with additional DPBS (0.8 mL). The purified ADCsolutions were further aliquoted for analytics and stored at 4° C.

Method G.

Conjugations were performed using a Tecan Freedom Evo robotic liquidhandling system. The solution of antibody (10 mg/mL) was preheated to37° C., and aliquoted onto a heated % deep-well plate in amounts of 3 mgper well (0.3 mL) and kept at 37 C. A solution of Bond-Breaker™tris(2-carboxyethyl)phosphine (TCEP) solution (1 mM, 0.051 mL, well) wasadded to antibodies, and the reaction mixture was kept at 37° C. for 75minutes. The solutions of reduced antibody were transferred to anunheated 96 deep-well plate. Corresponding solutions of synthons (5 mM,0.024 mL/well in DMSO) were added to the wells with reduced antibodiesfollowed by addition of boric buffer (pH=8, 0.03 mL % well) and treatedfor 3 days. The reaction solutions were loaded onto a platform (8×12) ofdesalting columns (NAP5, washed with DPBS 4× before use), followed byDPBS (0.3 mL) and eluted with additional DPBS (0.8 mL). The purified ADCsolutions were further aliquoted for analytics and stored at 4° C.

Method H.

A solution of Bond-Breaker™ tris(2-carboxyethyl)phosphine (TCEP)solution (10 mM, 0.17 mL) was added to the solution of antibody (10mg/mL, 10 mL) at room temperature. The reaction mixture was heated to37° C. for 75 minutes. The solution of synthon (10 mM, 0.40 mL in DMSO)was added to a solution of reduced antibody cooled to room temperature.The reaction solution was let to stand for 30 minutes at roomtemperature. The solution of ADC was treated with saturated ammoniumsulfate solution (˜2-2.5 mL) until a slightly cloudy solution formed.This solution was loaded onto butyl sepharose column (5 mL of butylsepharose) equilibrated with 30% phase B in phase A (phase A: 1.5 Mammonium sulphate. 25 mM phosphate; phase B: 25 mM phosphate. 25%isopropanol v/v). Individual fractions with DAR2 (also referred to as“E2”) and DAR4 (also referred to as “E4”) eluted upon applying gradientA/B up to 75% phase B. Each ADC solution was concentrated and bufferswitched using centrifuge concentrators or TFF for larger scales. Thepurified ADC solutions were filtered through a 0.2 micron, lowprotein-binding 13 mm syringe-filter and stored at 4° C.

Method I.

A solution of Bond-Breaker™ tris(2-carboxyethyl)phosphine (TCEP)solution (10 mM, 0.17 mL) was added to the solution of antibody (10mg/mL, 10 mL) at room temperature. The reaction mixture was heated to37° C. for 75 minutes. The solution of synthon (10 mM, 0.40 mL in DMSO)was added to a solution of reduced antibody cooled to room temperature.The reaction solution was let to stand for 30 minutes at roomtemperature. The solution of ADC was treated with saturated ammoniumsulfate solution (˜2-2.5 mL) until a slightly cloudy solution formed.This solution was loaded onto a butyl sepharose column (5 mL of butylsepharose) equilibrated with 30% phase B in Phase A (phase A: 1.5 Mammonium sulphate, 25 mM phosphate; phase B: 25 mM phosphate, 25%isopropanol v/v). Individual fractions with DAR2 (also referred to as“E2”) and DAR 4 (also referred to as “E4”) eluted upon applying agradient A/B up to 75% phase B. Each ADC solution was concentrated andbuffer switched using centrifuge concentrators or TFF for larger scales.The ADC solutions were treated with boric buffer (0.1 mL, 1M, pH8). Thereaction solution was let stand for 3 days at room temperature, thenloaded onto a desalting column (PD10, washed with DPBS 3×5 mL beforeuse), followed by DPBS (1.6 mL) and eluted with additional DPBS (3 mL).The purified ADC solution was filtered through a 0.2 micron, lowprotein-binding 13 mm syringe-filter and stored at 4° C.

DAR and Aggreeation of ADCs

The DAR and percentage aggregation of ADCs synthesized were determinedby LC-MS and size exclusion chromatography (SEC), respectively.

LC-MS General Methodology

LC-MS analysis was performed using an Agilent 1100 HPLC systeminterfaced to an Agilent LC/MSD TOF 6220 ESI mass spectrometer. The ADCwas reduced with 5 mM (final concentration) Bond-Breaker® TCEP solution(Thermo Scientific, Rockford, Ill.), loaded onto a Protein Microtrap(Michrom Bioresorces. Auburn. Calif.) desalting cartridge, and elutedwith a gradient of 10% B to 75% Bin 0.2 minutes at ambient temperature.Mobile phase A was H₂O with 0.1% formic acid (FA), mobile phase B wasacetonitrile with 0.1% FA, and the flow rate was 0.2 mL/min.Electrospray-ionization time-of-flight mass spectra of the co-elutinglight and heavy chains were acquired using Agilent MassHunter™acquisition software. The extracted intensity vs, m/z spectrum wasdeconvoluted using the Maximum Entropy feature of MassHunter software todetermine the mass of each reduced antibody fragment. DAR was calculatedfrom the deconvoluted spectrum by summing intensities of the naked andmodified peaks for the light chain and heavy chain normalized bymultiplying intensity by the number of drugs attached. The summed,normalized intensities were divided by the sum of the intensities, andthe summing results for two light chains and two heavy chains produced afinal average DAR value for the full ADC.

Thiosuccinimide hydrolysis of a bioconjugate can be monitored byelectrospray mass spectrometry, since the addition of water to theconjugate results in an increase of 18 Daltons to the observablemolecular weight of the conjugate. When a conjugate is prepared by fullyreducing the interchain disulfides of a human IgG1 antibody andconjugating the maleimide derivative to each of the resulting cysteines,each light chain of the antibody will contain a single maleimidemodification and each heavy chain will contain three maleimidemodifications, as described in FIG. 2. Upon complete hydrolysis of theresulting thiosuccinimides, the mass of the light chain will thereforeincrease by 18 Daltons, while the mass of each heavy chain will increaseby 54 Daltons. This is illustrated in FIG. 5 with the conjugation andsubsequent hydrolysis of an exemplary maleimide drug-linker (synthon TX,molecular weight 1736 Da) to the fully reduced huAb13v1 antibody.

Size Exclusion Chromatography General Methodology

Size exclusion chromatography was performed using a Shodex KW802.5column in 0.2 M potassium phosphate pH 6.2 with 0.25 mM potassiumchloride and 15% IPA at a flow rate of 0.75 ml/min. The peak areaabsorbance at 280 nm was determined for each of the high molecularweight and monomeric eluents by integration of the area under the curve.The % aggregate fraction of the conjugate sample was determined bydividing the peak area absorbance at 280 nM for the high molecularweight eluent by the sum of the peak area absorbances at 280 nM of thehigh molecular weight and monomeric eluents multiplied by 100%.

In Vitro Cell Viability Assay Methods

The tumor cell lines HCC38 (breast cancer), NCI-H1650 (NSCLC) andNCI-H847 (small cell lung cancer cell line) were obtained from AmericanType Culture Collection (ATCC). Cells were grown in 96-well cultureplates using recommended growth media overnight at a density of 5×10³(HCC38) or 20×10³ (NCI-H847) or 40×10³ (NCI-H1650) per well. Thefollowing day, treatments were added in fresh media to triplicate wells.Cellular viability was determined 5 days later using the CellTiter-GloLuminescent Cell Viability Assay kit (Promega), as directed in themanufacturer's protocol. Cell viability was assessed as percentage ofcontrol untreated cells.

Example 8: In vivo Efficacy of Anti-B7-H3 Antibody Drug Conjugates

Of the nine chimeric antibodies tested in vitro conjugated to CZsynthons, four showed subnanomolar cytotoxicity (Table 6), chAb3-CZ,chAb18-CZ, and chAb13-CZ achieved DARS ranging from 2.6 to 4.2 (seeTable 7) and were assessed for anti-tumor activity in a mouse small celllung cancer cell line xenograft model NCI-H146, of human origin, usingthe methods described below. Antibody MSL109 (an IgG1 antibody thatbinds to cytomegalovirus (CMV) glycoprotein H) was used as a control,both as a naked antibody and as an ADC (conjugated to the same synthon(CZ) as the chAb3, chAb18, and chAb13 antibodies). MSL109 is an isotypematched non-targeting control. The methods of this xenograft assay aredescribed below. The results are presented in Table 7. The results showthat each of the anti-B7-H3 Bcl-xL inhibiting ADCs were able tosignificantly inhibit tumor growth relative to the naked antibodycontrol (MSL109) or non-target specific Bcl-xL ADC control (MSL109-CZ).

TABLE 7 In vivo efficacy of chimeric anti-B7-H3 antibody as Bcl-xL drugconjugates Conjugation Dose^([a])/route/ Number ADC Method DAR regimenof mice TGI_(max) (%) TGD (%) MSL109 — — 10 mg/kg/IP/QD×1 8 0 0MSL109-CZ A 4.2 10 mg/kg/IP/QD×1 8 34 10 chAb3-CZ A 3.5 10 mg/kg/IP/QD×18 87 109 chAb18-CZ A 2.6 10 mg/kg/IP/QD×1 8 90 100 chAb13-CZ A 3.7 10mg/kg/IP/QD×1 8 81 104 ^([a])dose is given in mg/kg/day

Evaluation of Efficacy in Xenograft Models Methods

NCI-H146 cells, NCI-1650 cells, and EBC-1 cells were obtained from theAmerican Type Culture Collection (ATCC, Manassas. Va.). The cells werecultured as monolayers in RPMI-1640 (NCI-H146. NCI-H1650) or MEM (EBC-1)culture media (Invitrogen. Carlsbad, Calif.) that was supplemented with10% Fetal Bovine Serum (FBS, Hyclone, Logan, Utah). To generatexenografts, 5×10⁶ viable cells were inoculated subcutaneously into theright flank of immune deficient female SCID/bg mice (Charles RiverLaboratories, Wilmington, Mass.) respectively. The injection volume was0.2 mL and composed of a 1:1 mixture of S MEM and Matrigel (BD. FranklinLakes, N.J.). Tumors were size matched at approximately 200 mm³.Antibodies and conjugates were formulated in 0.9% sodium chloride forinjection and injected intraperitoneally. Injection volume did notexceed 200 μL. Therapy began within 24 hours after size matching of thetumors. Mice weighed approximately 22 g at the onset of therapy. Tumorvolume was estimated two to three times weekly. Measurements of thelength (L) and width (W) of the tumor were taken via electronic caliperand the volume was calculated according to the following equation:V=L×W²/2. Mice were euthanized when tumor volume reached 3,000 mm3 orskin ulcerations occurred. Eight mice were housed per cage. Food andwater were available ad libitum. Mice were acclimated to the animalfacilities for a period of at least one week prior to commencement ofexperiments. Animals were tested in the light phase of a 12-hour light:12-hour dark schedule (lights on at 06:00 hours). As described above,human IgG control antibody (MSL109) was used as a negative controlagent.

To refer to efficacy of therapeutic agents, parameters of amplitude(TGI_(max)), durability (TGD) of therapeutic response are used.TGI_(max) is the maximum tumor growth inhibition during the experiment.Tumor growth inhibition is calculated by 100*(1−T_(v)/C_(v)) where T_(v)and C_(v) are the mean tumor volumes of the treated and control groups,respectively. TGD or tumor growth delay is the extended time of atreated tumor needed to reach a volume of 1 cm relative to the controlgroup. TGD is calculated by 100*(T_(t)/C_(t)−r1) where T_(t) and C_(t)are the median time periods to reach 1 cm³ of the treated and controlgroups, respectively.

Example 9: Humanization of Anti-B7-H3 Antibody chAb18

Anti-B7-H3 chimeric antibody chAb18 was selected for humanization basedon its binding characteristics and favorable properties as an ADC,including its properties when conjugated to a Bel-xL inhibitor(described above as exemplary conjugate CZ).

Humanized antibodies were generated based on the variable heavy (VH) andvariable light (VL) CDR sequences of chAb18. Specifically, humangermline sequences were selected for constructing CDR-grafted, humanizedchAb18 antibodies, where the CDR domains of the VH and VL chains weregrafted onto different human heavy and light chain acceptor sequences.Based on the alignments with the VH and VL sequences of monoclonalantibody chAb18, the following human sequences were selected asacceptors:

-   -   IGHV1-69*06 and IGHJ6*01 for constructing heavy chain acceptor        sequences    -   IGKV1-9*01 and IGKJ2*01 for constructing light chain acceptor        sequences    -   IGKV6-21*01 and IGKJ2*01 as backup acceptor for constructing        light chain Thus, the VH and VL CDRs of chAb8 were grafted into        said acceptor sequences.

To generate humanized antibodies, framework back-mutations wereidentified and introduced into the CDR-grafted antibody sequences by denovo synthesis of the variable domain, or mutagenic oligonucleotideprimers and polymerase chain reactions, or both by methods well known inthe art. Different combinations of back mutations and other mutationswere constructed for each of the CDR-grafts as described below. Residuenumbers for these mutations are based on the Kabat numbering system.

For heavy chains huAb18VH.1, one or more of the following Vernier andVH/VL interfacing residues were back mutated as follows: L46P, L47W,G64V, F71H. Additional mutations include the following: Q1E, N60A, K64Q,D65G. For light chains huAb18VL.1, one or more of the following Vernierand VH/VL interfacing residues were back mutated as follows: A43S, L46P,L47W, G64V. G66V, F71H. For light chains huAb18VL.2, one or more of thefollowing Vernier and VH/VL interfacing residues were back mutated asfollows: L46P, L47W, K49Y. G64V, G66V, F71H.

The variable region and CDR amino acid sequences of the humanizedantibodies are described in Table 8 below.

TABLE 8 VH and VL amino acid sequences of humanized versions of chAb18SEQ ID Protein NO: Clone Region Residues Amino Acid Sequence 116huAb18VH.1 VH EVQLVQSGAEVKKPGSSVKVSCKAS GYSFTSYTIHWVRQAPGQGLEWMGYINPNSRNTDYNQKFKDRVTITADKS TSTAYMELSSLRSEDTAVYYCARYSGSTPYWYFDVWGQGTTVTVSS 25 huAb18VH.1 CDR-H1 Residues 26-35 GYSFTSYTIHof SEQ ID NO: 116 26 huAb18VH.1 CDR-H2 Residues 50-66 YINPNSRNTDYNQKFKDof SEQ ID NO: 116 27 huAb18VH.1 CDR-H3 Residues 99-110 YSGSTPYWYFDVof SEQ ID NO: 116 117 huAb18VH.1a VH EVQLVQSGAEVKKPGSSVKVSCKASGYSFTSYTIHWVRQAPGQGLEWIGY INPNSRNTDYNQKFKDRTTLTADRSTSTAYMELSSLRSEDTAVYYCARYS GSTPYWYFDVWGQGTTVTVSS 25 huAb18VH.1a CDR-H1Residues 26-35 GYSFTSYTIH of SEQ ID NO: 117 26 huAb18VH.1a CDR-H2Residues 50-66 YINPNSRNTDYNQKFKD of SEQ ID NO: 117 27 huAb18VH.1a CDR-H3Residues 99-110 YSGSTPYWYFDV of SEQ ID NO: 117 118 huAbl8VH.1b VHEVQLVQSGAEVKKPGSSVKVSCKAS GYSFTSYTIHWVRQAPGQGLEWMGYINPNSRNTDYAQKFQGRVTLTADKS TSTAYMELSSLRSEDTAVYYCARYSGSTPYWYFDVWGQGTTVTVSS 25 huAbl8VH.1b CDR-H1 Residues 26-35 GYSFTSYTIHof SEQ ID NO: 118 119 huAbl8VH.1b CDR-H2 Residues 50-66YINPNSRNTDYAQKFQG of SEQ ID NO: 118 27 huAbl8VH.1b CDR-H3Residues 99-110 YSGSTPYWYFDV of SEQ ID NO: 118 120 huAbl8VL.1 VLDIQLTQSPSFLSASVGDRVTITCRA SSSVSYMNWYQQKPGKAPKLLIYATSNLASGVPSRFSGSGSGTEFTLTIS SLQPEDFATYYCQQWSSNPLTFGQG TKLEIK 29 huAb18VL.1CDR-L1 Residues 24-33 RASSSVSYMN of SEQ ID NO: 120 30 huAb18VL.1 CDR-L2Residues 49-55 ATSNLAS of SEQ ID NO: 120 31 huAb18VL.1 CDR-L3Residues 88-96 QQWSSNPLT of SEQ ID NO: 120 121 huAb18VL.1a VLDIQLTQSPSFLSASVGDRVTITCRA SSSVSYMNWYQQKPGKSPKPWIYATSNLASGVPSRFSVSVSGTEHTLTIS SLQPEDFATYYCQQWSSNPLTFGQG TKLEIK 29hu18AbVL.1a CDR-L1 Residues 24-33 RASSSVSYMN of SEQ ID NO: 121 30huAb18VL.1a CDR-L2 Residues 49-55 ATSNLAS of SEQ ID NO: 121 31huAb18VL.1a CDR-L3 Residues 88-96 QQWSSNPLT of SEQ ID NO: 121 122huAb18VL.1b VL DIQLTQSPSFLSASVGDRVTITCRA SSSVSYMNWYQQKPGKAPKPWIYATSNLASGVPSRFSVSGSGTEHTLTIS SLQPEDFATYYCQQWSSNPLTFGQG TKLEIK 29huAb18VL.1b CDR-L1 Residues 24-33 RASSSVSYMN of SEQ ID NO: 122 30huAb18VL.1b CDR-L2 Residues 49-55 ATSNLAS of SEQ ID NO: 122 31huAb18VL.1b CDR-L3 Residues 88-96 QQWSSNPLT of SEQ ID NO: 122 123huAb18VL.2 VL EIVLTQSPDFQSVTPKEKVTITCRA SSSVSYMNWYQQKPDQSPKLLIKATSNLASGVPSRFSGSGSGTDFTLTIN SLEAEDAATYYCQQWSSNPLTFGQG TKLEIK 29 huAb18VL.2CDR-L1 Residues 24-33 RASSSVSYMN of SEQ ID NO: 123 30 huAb18VL.2 CDR-L2Residues 49-55 ATSNLAS of SEQ ID NO: 123 31 huAb18VL.2 CDR-L3Residues 88-96 QQWSSNPLT of SEQ ID NO: 123 124 huAb18VL.2a VLEIVLTQSPDFQSVTPKEKVTITCRA SSSVSYMNWYQQKPDQSPKPWIYATSNLASGVPSRFSVSVSGTDHTLTIN SLEAEDAATYYCQQWSSNPLTFGQG TKLEIK 29huAb18VL.2a CDR-L1 Residues 24-33 RASSSVSYMN of SEQ ID NO: 124 30huAbl8VL.2a CDR-L2 Residues 49-55 ATSNLAS of SEQ ID NO: 124 31huAb18VL.2a CDR-L3 Residues 88-96 QQWSSNPLT of SEQ ID NO: 124

Humanized variable regions of the murine monoclonal Ab18 (describedabove) were cloned into IgG expression vectors for functionalcharacterization:

-   -   Humanized Ab18VH.1 (huAb18VH.1) is a CDR-grafted, humanized Ab18        VH containing IGHV1-69*06 and IGHJ6*01 framework sequences. It        also contains a Q1E change to prevent pyroglutamate formation.        The variable and CDR sequences of huAb18VH.1 are described in        Table 8.    -   Humanized Ab18VHI.a (huAb18VH.1 a) is a humanized design based        on huAb18VH.1 and contains 4 proposed framework back-mutations:        M48I, V67T, L69I. K73R. The variable and CDR sequences of        huAb18VH.1 a are described in Table 8.    -   Humanized Ab18VH.b (huAb18VH.1b) is a humanized design based on        huAb18VH.1 and huAb18VH.1 a and contains 1 proposed framework        back-mutation L69I and 3 HCDR2 germlining changes N60A, K64Q,        D65G. The variable and CDR sequences of huAb18VH.1 b are        described in Table 8.    -   Humanized Ab18VL.1 (huAb18VL.1) is a CDR-grafted, humanized Ab18        VL containing IGKV1-9*01 and IGKJ2*01 framework sequences. The        variable and CDR sequences of huAb18VL.1 are described in Table        8.    -   Humanized Ab18VL.1a (huAb18VL.1a) is a humanized design based on        huAb18VL.1 and contains 6 proposed framework back-mutations:        A43S. L46P, L47W, G64V, G66V, F71H. The variable and CDR        sequences of huAb18VL.11 are described in Table 8. Humanized        Ab18VL.1b (huAb18VL.1b) is a humanized design based on        huAb18VL.1 and huAb18VL.1a contains 4 proposed framework        back-mutations: L46P. L47W, G64V, F71H. The variable and CDR        sequences of huAb18VL.1b are described in Table 8.    -   Humanized Ab18VL.2 (huAb18VL.2) is a CDR-grafted, humanized Ab18        VL containing IGKV6-21*01 and IGKJ2*01 framework sequences. The        variable and CDR sequences of huAb18VL.2 are described in Table        8.    -   Humanized Ab18VL.2a (huAb18VL.2a) is a humanized design based on        huAb18VL.2 and contains 6 proposed framework back-mutations:        L46P. L47W, K49Y, G64V, G66V, F71H. The variable and CDR        sequences of huAb18VL.2a are described in Table 8.

Thus, the humanization of chAb18 resulted in 10 humanized antibodies,including huAb18v1, huAb18v2, huAb18v3, huAb18v4, huAb18v5, huAb18v6,huAb18v7, huAb18v8, huAb18v9, and huAb18v10. The variable and heavylight chains for each of these humanized versions of Ab18 are providedbelow:

TABLE 9 Anti-B7-H3 Ab18 humanized antibodies huAb18v1 huAb18VH1/huAb18VL1 huAb18v2 huAb18 VH1b/huAb18VL1 huAb18v3 huAb18VH1a/huAbVL1a huAb18v4 huAb18 VH1b/huAb18VL1a huAb18v5 huAn18VH1/huAb18VL2 huAb18v6 huAb18 VH1b/huAb18VL2 huAb18v7 huAb18 VH1b/huAb18VL2a huAb18v8 huAb18 VH1a/huAb18 VL1b huAb18v9 huAb18 VH1a/huAb18 VL2ahuAb18v10 huAb18 VH1b/huAb18 VL1b

Example 10: In vitro Characterization of Anti-B7-H3 chAb18 HumanizedVariants

The humanization of chAb18 generated 10 variants (described above inTable 9) that retained binding to human and cyno B7-H3 as assessed byFACS (the method of which is described above in Example 6). Thesevariants were further characterized for binding by SPR and weresuccessfully conjugated to the Bcl-xL inhibitor synthon CZ using MethodA (described above) and assessed for cell cytotoxicity as described inExample 7. Table 10 summarizes the in vitro characteristics of thevarious humanized Ab18 variants. The parental chAb18 from which thevariants were drivce was also tested as a comparator. All humanizedvariants had similar binding properties as assessed by biacore,andretained binding activity t cell surface expressed as conjugates withthe CZ synthon. The cytotoxicity of all of the variants as CZ synthonswere similar to the chAb18S from which they were derived.

TABLE 10 In vitro characterization of humanized anti-B7-H3 Ab18 variantsAffinity of FACS naked DAR Binding mAbs Cytotoxicity Sequence by to huB7- (Biacore, (HCC38 Cell Variant name Numbers MS % agg by SEC H3)K_(D)) line IC₅₀) chAb18-CZ 24, 28 2.3 1.14 1.70E−10 0.28 huAb18v1-CZ116, 120 2.6 3.3 1.27 5.20E−10 0.39 huAb18v2-CZ 118, 120 1.8 3.3 2.256.90E−10 1.19 huAb18v3-CZ 117, 121 2.4 3.6 1.27 2.30E−10 0.32huAb18v4-CZ 118, 121 2.5 5.5 0.90 5.70E−10 0.29 huAb18v5-CZ 116, 123 3.44.2 2.91 2.30E−10 0.12 huAb18v6-CZ 118, 123 3.4 3.5 2.09 2.00E−10 0.14huAb18v7-CZ 118, 124 4.3 3.6 1.92 4.00E−10 0.03 huAb18v8-CZ 117, 122 2.63.5 1.98 2.50E−10 1.3 huAb18v9-CZ 117, 124 2.4 3.8 1.58 3.80E−10 0.9huAb18v10-CZ 118, 122 2.7 3.2 1.19 2.50E−10 0.57

Humanized chAb18 variants were conjugated to the CZ synthon and testedfor cytotoxicity in HCC38 cell line. As described in Table 10, mosthumanized antibodies showed potent cytotoxicity, similar to thoseobserved with control antibody chAb18.

Example 11: In Vivo Efficacy of Humanized Ab18 Variants as Bcl-xLInhibitor ADCs

Six of the humanized chAb18 variants were selected based on in vitrocytotoxicity results described in Example 10. Specifically, antibodieshuAb18v1, huAb18v3, huAb18v4, huAb18v6, huAb18v7, and huAb18v9 were eachconjugated to the CZ synthon (to form an anti-B7-H3 CZ ADC) forevaluation in an in vivo xenograft model of small cell lung cancer(using NCI-H146 cells), as described in Example 8. Single dose treatmentof the tumor bearing mice resulted in tumor growth inhibition and tumorgrowth delay and the results are summarized in Table 11. Ab095 was usedas a negative control for the effect of administering IgG, as it is anisotype matched non-target specific antibody raised against tetanustoxoid. See Larrick et al., 1992, Immunological Reviews 69-85. Mice wereadministered 6 mg/kg of the ADC intraperitoneally QDx1.

TABLE 11 In vivo efficacy of anti-B7-H3 ADCS (humanized chAb18-CZvariants) Conjugation DAR by Dose^([a])//route/ Number of TGI_(max) ADCMethod MS regimen mice (%) TGD (%) AB095 — n/a 6 mg/kg/IP/QDx1 8 0 0huAb18v1-CZ A 2.6 6 mg/kg/IP/QDx1 8 79 45 huAb18v3-CZ A 2.4 6mg/kg/IP/QDx1 8 81 39 huAB18v4-CZ A 2.5 6 mg/kg/IP/QDx1 8 85 48huAB18v6-CZ A 3.4 6 mg/kg/IP/QDx1 8 86 45 huAb18v7-CZ A 4.3 6mg/kg/IP/QDx1 8 87 42 huAb18v9-CZ A 2.4 6 mg/kg/IP/QDx1 8 83 35^([a])dose is given in mg/kg/day

As described in Table 11, each of the tested humanized antibodies wasable to inhibit tumor growth in the mouse xenograf model.

Example 12: Humanization of Anti-B7-H3 Antibody chAb3

Anti-B7-H3 chimeric antibody chAb3 was selected for humanization basedon its favorable properties as a Bcl-xL inhibiting (Bcl-xLi) conjugate.Humanized antibodies were generated based on the variable heavy (VH) andvariable light (VL) CDR sequences of chAB3. Specifically, human germlinesequences were selected for constructing CDR-grafted, humanized chAb3antibodies where the CDR domains of the VH and VL chains of chAb3 weregrafted onto different human heavy and light chain acceptor sequences.Based on the alignments with the VH and VL sequences of monoclonalantibody chAb3 the following human sequences were selected as acceptors:

-   -   IGHV1-69*06 and IGHJ6*01 for constructing heavy chain acceptor        sequences    -   IGKV2-28*01 and IGKJ4*01 for constructing light chain acceptor        sequences PGP425,DNA

IGHV1-69*06 IGHJ6 (SEQ ID NO: 174)QVQLVQSGAEVKKPGSSVKVSCKASggtfssyaisWVRQAPGQGLEWMGgiipifgtanyaqkfqgRVTITADKSTSTAYMELSSLRSEDTAVYYCARxx xxxxxxWGQGTTVTVSS;where xxxxxxxx represents the CDR-H3 region. IGKV2-28*01 IGKJ4(SEQ ID NO: 175) DIVMTQSPLSLPVTPGEPASISCrssqsllhsngynyldWYLQKPGQSPQLLIYlgsnrasGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCxxxxxxx xxFGGGTKVEIK;where xxxxxxxxx represents the CDR-L3 region.

By grafting the corresponding VH and VL CDRs of chAb3 into said acceptorsequences, CDR-grafted, humanized, and modified VH and VL sequences wereprepared. To generate humanized antibodies with potential frameworkback-mutations, the mutations were identified and introduced into theCDR-grafted antibody sequences by de novo synthesis of the variabledomain, or mutagenic oligonucleotide primers and polymerase chainreactions, or both. Different combinations of back mutations and othermutations are constructed for each of the CDR-grafts as follows. Residuenumbers for these mutations are based on the Kabat numbering system.

The amino acid sequences of the various humanized heavy and light chainvariable regions are described below in Table 12.

For heavy chains huAb3VH.1, one or more of the following Vernier andVH/VL interfacing residues were back mutated as follows: M48I, V67A,I69L, A71V, K73R, M80V, Y91F, R94G. For light chains huAb31 VL.1, one ormore of the following Vernier and VH/VL interfacing residues were backmutated as follows: I2V, Y87F.

The following humanized variable regions of the murine monoclonal chAb3antibody were cloned into IgG expression vectors for functionalcharacterization:

-   -   Humanized Ab3 VH.1 (huAb3VH.1) is a CDR-grafted, humanized Ab3        VH containing IGHV1-69*06 and IGHJ6*01 framework sequences. It        also contains a Q1E change to prevent pyroglutamate formation.    -   Humanized Ab3 VH.1a (huAb3VH.1a) is a humanized design based on        huAb3VH.1 and contains 8 proposed framework back-mutations:        M48I, V67A, I69L, A71V, K73R, M80V, Y91F, R94G.    -   Humanized Ab3 VH.1b (huAb3VH.1b) is a humanized design between        huAb3VH.1 and huAb3VH.1a and contains 6 proposed framework        back-mutations: M48I, V67A, I69L, A71V. K73R, R94G.    -   Humanized Ab3 VL.1 (huAb3VL.1) is a CDR-grafted, humanized Ab3        VL containing IGKV2-28*01 and IGKJ4*01 framework sequences.    -   Humanized Ab3 VL.1a (huAb3VL.1a is a humanized design based on        huAb3VL.1 and contains 2 proposed framework back-mutations: I2V,        Y87F.    -   Humanized Ab3 VL.1b (huAb3VL.1b) is a humanized design contains        only 1 proposed framework back-mutations: I2V.

The variable region and CDR amino acid sequences of the foregoinghumanized antibodies are described in Table 12 below.

TABLE 12 VH and VL sequences of humanized versions of chAb3 SEQ IDProtein NO: Clone Region Residues Amino Acid Sequence 125 huAb3VH.1 VHEVQLVQSGAEVKKPGSSVKVS CKASGYTFSSYWMHWVRQAPG QGLEWMGLIHPDSGSTNYNEMFKNRVTITADKSTSTAYMELS SLRSEDTAVYYCARGGRLYFD YWGQGTTVTVSS 10 huAb3VH.1CDR-H1 Residues 26-35 GYTFSSYWMH of SEQ ID NO: 125 11 huAb3VH.1 CDR-H2Residues 50-66 LIHPDSGSTNYNEMFKR of SEQ ID NO: 125 12 huAb3VH.1 CDR-H3Residues 99-106 GGRLYFDY of SEQ ID NO: 125 126 huAb3VH.1a VHEVQLVQSGAEVKKPGSSVKVS CKASGYTFSSYWMHWVRQAPG QGLEWIGLIHPDSGSTNYNEMFKNRATLTVDRSTSTAYVELS SLRSEDTAVYFCAGGGRLYFD YWGQGTTVTVSS 10 huAb3VH.1aCDR-H1 Residues 26-35 GYTFSSYWMH of SEQ ID NO: 126 11 huAb3VH.1a CDR-H2Residues 50-66 LIHPDSGSTNYNEMFKR of SEQ ID NO: 126 12 huAb3VH.1a CDR-H3Residues 99-106 GGRLYFDY of SEQ ID NO: 126 127 huAb3VH.1b VHEVQLVQSGAEVKKPGSSVKVS CKASGYTFSSYWMHWVRQAPG QGLEWIGLIHPDSGSTNYNEMFKNRATLTVDRSTSTAYMELS SLRSEDTAVYYCAGGGRLYFD YWGQGTTVTVSS 10 huAb3VH.1bCDR-H1 Residues 26-35 GYTFSSYWMH of SEQ ID NO: 127 11 huAb3VH.1b CDR-H2Residues 50-66 LIHPDSGSTNYNEMFKR of SEQ ID NO: 127 12 huAb3VH.1b CDR-H3Residues 99-106 GGRLYFDY of SEQ ID NO: 127 128 huAb3VL.1 VLDIVMTQSPLSLPVTPGEPASI SCRSSQSLVHSNGDTYLRWYL QKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVE AEDVGVYYCSQSTHVPYTFGG GTKVEIK 14 huAb3VL.1 CDR-L1Residues 24-39 RSSQSLVHSNGDTYLR of SEQ ID NO: 128 7 huAb3VL.1 CDR-L2Residues 55-61 KVSNRFS of SEQ ID NO: 128 15 huAb3VL.1 CDR-L3Residues 94-102 SQSTHVPYT of SEQ ID NO: 128 129 huAb3VL.1a VLDVVMTQSPLSLPVTPGEPASI SCRSSQSLVHSNGDTYLRWYL QKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVE AEDVGVYFCSQSTHVPYTFGG GTKVEIK 14 huAb3VL.1a CDR-L1Residues 24-39 RSSQSLVHSNGDTYLR of SEQ ID NO: 129 7 huAb3VL.1a CDR-L2Residues 55-61 KVSNRFS of SEQ ID NO: 129 15 huAb3VL.1a CDR-L3Residues 94-102 SQSTHVPYT of SEQ ID NO: 129 130 huAb3VL.1b VLDVVMTQSPLSLPVTPGEPASI SCRSSQSLVHSNGDTYLRWYL QKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVE AEDVGVYYCSQSTHVPYTFGG GTKVEIK 14 huAb3VL.1b CDR-L1Residues 24-39 RSSQSLVHSNGDTYLR of SEQ ID NO: 130 7 huAb3VL.1b CDR-L2Residues 55-61 KVSNRFS of SEQ ID NO: 130 15 huAb3VL.1b CDR-L3Residues 94-102 SQSTHVPYT of SEQ ID NO: 130

The humanization of chAb3 resulted in 6 humanized antibodies, includinghuAb3v1, huAb3v2 huAb3v3, huAb3v4, huAb18v5, and huAb3v6. The variableand heavy light chains for each of these humanized versions of Ab18 areprovided below in Table 13.

TABLE 13 Humanized Ab3 antibodies huAb3v1 huAb3 VH1/huAb3 VL1 huAb3v2huAb3 VH1b/huAb3 VL1 huAb3v3 huAb3 VH1a/huAb3 VL1a huAb3v4 huAb3VH1/huAb3 VL1b huAb3v5 huAb3 VH1b/huAb3 VL1b huAb3v6 huAb3 VH1a/huAb3VL1b

Example 13: In Vitro Characterization of chAb3 Humanized Variants

The humanization of chAb3 generated 6 variants (described in Table 13)that retained binding to human B7-H3 as assessed by FACS (as describedin Example 6). These variants were further characterized for binding bySPR and as ADCs conjugated to the Bcl-xL inhibitor synthon (linkerwarhead) CZ. The humanized Ab3 antibodies were also assessed for cellcytotoxicity (using the assay described above in Example 7). Table 14summarizes in vitro characteristics of chAb3 humanized variants. An ADCcomprising chAb3 conjugated to synthon CZ was used as a control.

TABLE 14 In vitro characterization of humanized variants of chAb3 FACSCytotoxicity DAR Binding to Affinity of (HCC38 Cell Seq. Id. Conjugationby % agg hu B7-H3) naked mAbs line IC₅₀) ADC Number Method MS by SECEC₅₀ (nM) (Biacore, K_(D)) (nM) chAb3-CZ  9, 13 A 3.8 0.61 1.90E−08 0.17huAb3v1- 125, 128 A 3.6 3.3 1.45 5.20E−10 0.53 CZ huAb3v2- 127, 128 A3.8 10.1 0.73 6.90E−10 0.13 CZ huAb3v3- 126, 129 A 3.6 2.5 1.68 2.30E−109.22 CZ huAb3v4- 125, 130 A 3.1 3.1 n/a 5.70E−10 n/a CZ huAb3v5- 127,130 A 3.1 5.9 0.85 2.30E−10 0.17 CZ huAb3v6- 126, 130 A 3.3 4.9 1.782.00E−10 0.13 CZ

Example 14: In vivo Efficacy of chAb3 Humanized Variants as Bc-xL ADCs

Two of the humanized variants (huAb3v2 and huAb3v6) were selected basedon potent in vitro cytotoxicity as CZ conjugates and acceptableaggregation properties for evaluation in an in vivo murine xenograftmodel of small cell lung cancer cells (NCI-H146 cells) as described inmaterials and methods in Example 8. Single dose treatment of tumorbearing mice resulted in tumor growth inhibition and tumor growth delayfor both humanized antibodies conjugated to an exemplary Bcl-xLinhibitor, and the results are summarized in Table 15.

TABLE 15 In vivo efficacy of humanized chAb3-CZ variants ConjugationDose^([a])/route/ Number of ADC Method DAR regimen mice TGI_(max) (%)TGD (%) AB095 — 6 mg/kg/IP/QDx1 8 0 0 huAb3v2-CZ A 3.8 6 mg/kg/IP/QDx1 883 52 huAb3v6-CZ A 3.3 6 mg/kg/IP/QDx1 8 91 88 ^([a])dose is given inmg/kg/day

Example 15: Modifications of the CDRs of Humanized Variant AntibodyhuAb3v2

huAb3v2 showed favorable binding and cell killing properties. Anexamination of the variable region amino acid sequences of huAb3v2,however, revealed potential deamidation and/or isomerization sites.

The amino acid sequences of huAb3 variable regions are described below,including the light chain (huAb3VL1) and the heavy chain (huAb3VH1b).The potential deamidation and/or isomerization sites in CDRs of the VH(CDR2 at amino acids “ds” and VL (CDR1 at amino acids “ng”) areitalicized and were thus engineered to improve antibody manufacturing.The CDRs are described in lower case letters in the sequences below.

To make huAb3v2 variants lacking these potential deamidation and/orisomerization sites, each of the amino acids indicated below (x and z;representing the potential sites in the CDR1 of the VL and the CDR2 ofthe VH) were mutagenized. The resulting 30 VL variants were paired withthe original huAb3v2 VH and tested for binding. The resulting 29 VHvariants were paired with the original huAb3v2 VL and tested forbinding. Successful VH variants were combined and tested with productiveVL variants harboring changes in LCDR1 to make the final humanizedvariants lacking the potential deamidation and/or isomerization sites inCDRs. The amino acid sequences of the variants are provided in Table 16below. The full length amino acid sequences of the heavy chain and lightchain of the huAb3v2 variant, huAb3v2.5 are provided in SEQ ID NOs: 170and 171, respectively. The full length amino acid sequences of the heavychain and light chain of the huAb3v2 variant, huAb3v2.6 are provided inSEQ ID NOs: 172 and 173, respectively.

huAb3 VL1 (SEQ ID NO: 128) DIVMTQSPLSLPVTPGEPASISCrssqslvhs

dtylrWYLQKPGQSPQ LLIYkvsnrfsGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCsqsthvpytFGGGTKVEIK (SEQ ID NO: 178)

g (15 variants)  (SEQ ID NO: 179) n

 (15 variants)  huAb3 VH1b (SEQ ID NO: 127)EVQLVQSGAEVKKPGSSVKVSCKASgytfssywmhWVRQAPGQGLEWIGl ihp

gstnynemfknRATLTVDRSTSTAYMELSSLRSEDTAVYYCAGgg rlyfdyWGQGTTVTVSS(SEQ ID NO: 180) (15 variants) 

s (SEQ ID NO: 181) (14 variants) d

where (for both the VL and VH),

x=All amino acids, except: M, C, N, D, or Q.

z=All amino acids, except: M, C, G, S, N, or P.

Proposed framework back mutations are underlined (see Example 12).

TABLE 16 Variable region sequences of huAb3v2 antibody variants SEQ IDProtein NO: Clone Region Residues Amino Acid Sequence 131 huAb3v2.1 VHEVQLVQSGAEVKKPGSSVKVSCK ASGYTFSSYWMHWVRQAPGQGLE WIGLIHPWSGSTNYNEMFKNRATLTVDRSTSTAYMELSSLRSEDTA VYYCAGGGRLYFDYWGQGTTVTV SS 10 huAb3v2.1 CDR-H1Residues 26-35 GYTFSSYWMH of SEQ ID NO: 131 132 huAb3v2.1 CDR-H2Residues 50-66 LIHPWSGSTNYNEMFKN of SEQ ID NO: 131 12 huAb3v2.1 CDR-H3Residues 99-106 GGRLYFDY of SEQ ID NO: 131 133 huAb3v2.1 VLDIVMTQSPLSLPVTPGEPASISC RSSQSLVHSSGDTYLRWYLQKPG QSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY CSQSTHVPYTFGGGTKVEIK 134 huAb3v2.1 CDR-L1Residues 24-39 RSSQSLVHSSGDTYLR of SEQ ID NO: 133 7 huAb3v2.1 CDR-L2Residues 55-61 KVSNRFS of SEQ ID NO: 133 15 huAb3v2.1 CDR-L3Residues 94-102 SQSTHVPYT of SEQ ID NO: 133 131 huAb3v2.2 VHEVQLVQSGAEVKKPGSSVKVSCK ASGYTFSSYWMHWVRQAPGQGLE WIGLIHPWSGSTNYNEMFKNRATLTVDRSTSTAYMELSSLRSEDTA VYYCAGGGRLYFDYWGQGTTVTV SS 10 huAb3v2.2 CDR-H1Residues 26-35 GYTFSSYWMH of SEQ ID NO: 131 132 huAb3v2.2 CDR-H2Residues 50-66 LIHPWSGSTNYNEMFKN of SEQ ID NO: 131 12 huAb3v2.2 CDR-H3Residues 99-106 GGRLYFDY of SEQ ID NO: 131 135 huAb3v2.2 VLDIVMTQSPLSLPVTPGEPASISC RSSQSLVHSNRDTYLRWYLQKPG QSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY CSQSTHVPYTFGGGTKVEIK 136 huAb3v2.2 CDR-L1Residues 24-39 RSSQSLVHSNRDTYLR of SEQ ID NO: 135 7 huAb3v2.2 CDR-L2Residues 55-61 KVSNRFS of SEQ ID NO: 135 15 huAb3v2.2 CDR-L3Residues 94-102 SQSTHVPYT of SEQ ID NO: 135 131 huAb3v2.3 VHEVQLVQSGAEVKKPGSSVKVSCK ASGYTFSSYWMHWVRQAPGQGLE WIGLIHPWSGSTNYNEMFKNRATLTVDRSTSTAYMELSSLRSEDTA VYYCAGGGRLYFDYWGQGTTVTV SS 10 huAb3v2.3 CDR-H1Residues 26-35 GYTFSSYWMH of SEQ ID NO: 131 132 huAb3v2.3 CDR-H2Residues 50-66 LIHPWSGSTNYNEMFKN of SEQ ID NO: 131 12 huAb3v2.3 CDR-H3Residues 99-106 GGRLYFDY of SEQ ID NO: 131 137 huAb3v2.3 VLDIVMTQSPLSLPVTPGEPASISC RSSQSLVHSNQDTYLRWYLQKPG QSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY CSQSTHVPYTFGGGTKVEIK 138 huAb3v2.3 CDR-L1Residues 24-39 RSSQSLVHSNQDTYLR of SEQ ID NO: 137 7 huAb3v2.3 CDR-L2Residues 55-61 KVSNRFS of SEQ ID NO: 137 15 huAb3v2.3 CDR-L3Residues 94-102 SQSTHVPYT of SEQ ID NO: 137 139 huAb3v2.4 VHEVQLVQSGAEVKKPGSSVKVSCK ASGYTFSSYWMHWVRQAPGQGLE WIGLIHPESGSTNYNEMFKNRATLTVDRSTSTAYMELSSLRSEDTA VYYCAGGGRLYFDYWGQGTTVTV SS 10 huAb3v2.4 CDR-H1Residues 26-35 GYTFSSYWMH of SEQ ID NO: 139 140 huAb3v2.4 CDR-H2Residues 50-66 LIHPESGSTNYNEMFKN of SEQ ID NO: 139 12 huAb3v2.4 CDR-H3Residues 99-106 GGRLYFDY of SEQ ID NO: 139 133 huAb3v2.4 VLDIVMTQSPLSLPVTPGEPASISC RSSQSLVHSSGDTYLRWYLQKPG QSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY CSQSTHVPYTFGGGTKVEIK 134 huAb3v2.4 CDR-L1Residues 24-39 RSSQSLVHSSGDTYLR of SEQ ID NO: 133 7 huAb3v2.4 CDR-L2Residues 55-61 KVSNRFS of SEQ ID NO: 133 15 huAb3v2.4 CDR-L3Residues 94-102 SQSTHVPYT of SEQ ID NO: 133 139 huAb3v2.5 VHEVQLVQSGAEVKKPGSSVKVSCK ASGYTFSSYWMHWVRQAPGQGLE WIGLIHPESGSTNYNEMFKNRATLTVDRSTSTAYMELSSLRSEDTA VYYCAGGGRLYFDYWGQGTTVTV SS 10 huAb3v2.5 CDR-H1Residues 26-35 GYTFSSYWMH of SEQ ID NO: 139 140 huAb3v2.5 CDR-H2Residues 50-66 LIHPESGSTNYNEMFKN of SEQ ID NO: 139 12 huAb3v2.5 CDR-H3Residues 99-106 GGRLYFDY of SEQ ID NO: 139 135 huAb3v2.5 VLDIVMTQSPLSLPVTPGEPASISC RSSQSLVHSNRDTYLRWYLQKPG QSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY CSQSTHVPYTFGGGTKVEIK 136 huAb3v2.5 CDR-L1Residues 24-39 RSSQSLVHSNRDTYLR of SEQ ID NO: 135 7 huAb3v2.5 CDR-L2Residues 55-61 KVSNRFS of SEQ ID NO: 135 15 huAb3v2.5 CDR-L3Residues 94-102 SQSTHVPYT of SEQ ID NO: 135 139 huAb3v2.6 VHEVQLVQSGAEVKKPGSSVKVSCK ASGYTFSSYWMHWVRQAPGQGLE WIGLIHPESGSTNYNEMFKNRATLTVDRSTSTAYMELSSLRSEDTA VYYCAGGGRLYFDYWGQGTTVTV SS 10 huAb3v2.6 CDR-H1Residues 26-35 GYTFSSYWMH of SEQ ID NO: 139 140 huAb3v2.6 CDR-H2Residues 50-66 LIHPESGSTNYNEMFKN of SEQ ID NO: 139 12 huAb3v2.6 CDR-H3Residues 99-106 GGRLYFDY of SEQ ID NO: 139 137 huAb3v2.6 VLDIVMTQSPLSLPVTPGEPASISC RSSQSLVHSNQDTYLRWYLQKPG QSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY CSQSTHVPYTFGGGTKVEIK 138 huAb3v2.6 CDR-L1Residues 24-39 RSSQSLVHSNQDTYLR of SEQ ID NO: 137 7 huAb3v2.6 CDR-L2Residues 55-61 KVSNRFS of SEQ ID NO: 137 15 huAb3v2.6 CDR-L3Residues 94-102 SQSTHVPYT of SEQ ID NO: 137 141 huAb3v2.7EVQLVQSGAEVKKPGSSVKVSCK ASGYTFSSYWMHWVRQAPGQGLE WIGLIHPISGSTNYNEMFKNRATLTVDRSTSTAYMELSSLRSEDTA VYYCAGGGRLYFDYWGQGTTVTV SS 10 huAb3v2.7 CDR-H1Residues 26-35 GYTFSSYWMH of SEQ ID NO: 141 142 huAb3v2.7 CDR-H2Residues 50-66 LIHPISGSTNYNEMFKN of SEQ ID NO: 141 12 huAb3v2.7 CDR-H3Residues 99-106 GGRLYFDY of SEQ ID NO: 141 133 huAb3v2.7DIVMTQSPLSLPVTPGEPASISC RSSQSLVHSSGDTYLRWYLQKPG QSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY CSQSTHVPYTFGGGTKVEIK 134 huAb3v2.7 CDR-L1Residues 24-39 RSSQSLVHSSGDTYLR of SEQ ID NO: 133 7 huAb3v2.7 CDR-L2Residues 55-61 KVSNRFS of SEQ ID NO: 133 15 huAb3v2.7 CDR-L3Residues 94-102 SQSTHVPYT of SEQ ID NO: 133 141 huAb3v2.8 VHEVQLVQSGAEVKKPGSSVKVSCK ASGYTFSSYWMHWVRQAPGQGLE WIGLIHPISGSTNYNEMFKNRATLTVDRSTSTAYMELSSLRSEDTA VYYCAGGGRLYFDYWGQGTTVTV SS 10 huAb3v2.8 CDR-H1Residues 26-35 GYTFSSYWMH of SEQ ID NO: 141 142 huAb3v2.8 CDR-H2Residues 50-66 LIHPISGSTNYNEMFKN of SEQ ID NO: 141 12 huAb3v2.8 CDR-H3Residues 99-106 GGRLYFDY of SEQ ID NO: 141 135 huAb3v2.8 VLDIVMTQSPLSLPVTPGEPASISC RSSQSLVHSNRDTYLRWYLQKPG QSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY CSQSTHVPYTFGGGTKVEIK 136 huAb3v2.8 CDR-L1Residues 24-39 RSSQSLVHSNRDTYLR of SEQ ID NO: 135 7 huAb3v2.8 CDR-L2Residues 55-61 KVSNRFS of SEQ ID NO: 135 15 huAb3v2.8 CDR-L3Residues 94-102 SQSTHVPYT of SEQ ID NO: 135 141 huAb3v2.9 VHEVQLVQSGAEVKKPGSSVKVSCK ASGYTFSSYWMHWVRQAPGQGLE WIGLIHPISGSTNYNEMFKNRATLTVDRSTSTAYMELSSLRSEDTA VYYCAGGGRLYFDYWGQGTTVTV SS 10 huAb3v2.9 CDR-H1Residues 26-35 GYTFSSYWME of SEQ ID NO: 141 142 huAb3v2.9 CDR-H2Residues 50-66 LIHPISGSTNYNEMFKN of SEQ ID NO: 141 12 huAb3v2.9 CDR-H3Residues 99-106 GGRLYFDY of SEQ ID NO: 141 137 huAb3v2.9 VLDIVMTQSPLSLPVTPGEPASISC RSSQSLVHSNQDTYLRWYLQKPG QSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY CSQSTHVPYTFGGGTKVEIK 138 huAb3v2.9 CDR-L1Residues 24-39 RSSQSLVHSNQDTYLR of SEQ ID NO: 137 7 huAb3v2.9 CDR-L2Residues 55-61 KVSNRFS of SEQ ID NO: 137 15 huAb3v2.9 CDR-L3Residues 94-102 SQSTHVPYT of SEQ ID NO: 137

Example 16: In Vitro Characterization of huAb3v2 Variants

Removal of potential deamidation and/or isomerization sites (describedin Example 15) generated only 6 variants that retained binding to bothhuman and cyno B7-H3 exogenously expressed on mouse 3T12 fibroblasts asassessed by FACS (as described in the methods of Example 6).

These new anti-B7-1H-3antibodies were further characterized for bindingby SPR and conjugated to the Bel-xLi synthon CZ and assessed for cellcytotoxicity (using the methods described in Example 7). Table 17provides in vitro characteristics of six huAb3v2 humanized variants.

TABLE 17 In vitro characterization of humanized hu,kb3 v2 variants,including naked antibodies and ADCs Affinity of Cyto- % FACS nakedtoxicity agg ELISA (EC₅₀ nM) mAbs (H847 Cell Sequence Conjugation DAR byhB7-H3 hB7- cyB7- (Biacore, line IC₅₀) ADC number Method by MS SEC EC₅₀nM H3 H3 KD) (nM) huAb3v2- 127, 128 A 3.5 0.44 5.11 2.87 2.30E−09 1.49CZ huAb3v2.2- 131, 135 A 0.7 1.8 0.10 5.29 3.68 Poor fit 26.7 CZhuAb3v2.3- 131, 137 A 1.1 1.5 0.11 6.50 4.03 Poor fit — CZ huAb3v2.5-139, 135 A 3.4 15.6 0.13 5.14 4.86 5.30E−09 1.57 CZ huAb3v2.6- 139, 137A 3.3 15 0.09 5.64 3.31 5.80E−08 1.70 CZ huAb3v2.8- 141, 135 A 2.0 5.70.14 3.94 3.01 Poor fit 2.36 CZ huAb3v2.9- 141, 137 A 2.7 4.3 0.16 6.164.64 Poor fit 2.30 CZ

As described in Table 17, the results showed that all six huAb3v2variants had similar binding properties to cells expressing human orcynoB7-H3 as compared to the parental huAb3v2. Of the six huAb3v2variants, four antibodies (huAb3v2.5, huAb3v2.6, huAb3v2.8, huAb3v2.9)showed potent cytotoxicity in H847 cells when conjugated to exemplaryBcl-xLi synthon CZ.

Example 17: Humanization of Anti-B7-H3 Antibody chAb13

The anti-B7-H3 chimeric antibody chAb13 was selected for humanizationbased on its binding characteristics and favorable properties as an ADC(conjugated to a Bcl-xL inhibitor).

Prior to humanization, chAb13 was modified in order to minimizepotential deamidation in the light chain CDR3 (QQYNSYPFT (SEQ IDNO:182), potential deamidation site is indicated as residues “NS”(italicized)). Point mutations in the amino acid position correspondingto “N” and/or “S” within the light chain CDR3 of chAb13 were introduced,resulting in 30 variants. Antibodies containing these CDR3 light chainvariants were then screened for their ability to retain the bindingcharacteristics of chAb13. Variants comprising a CDR3 having atryptophan (W) point mutation instead of the serine “S” in the “NS”motif (i.e., QQYNWYPFT (SEQ ID NO: 39)) retained the binding features ofthe parent chAb13 antibody. The substitution of the S residue with a Wresidue within the CDR3 was surprising given the structural differencesbetween serine and tryptophan as well as the significant role the CDR3plays in antigen binding.

Humanized antibodies were generated based on the variable heavy (VH) andvariable light (VL) CDR sequences of chAb13, including the “NW” lightchain CDR3. Specifically, human germline sequences were selected forconstructing CDR-grafted, humanized chAb13 antibodies, where the CDRdomains of the VH and VL chains were grafted onto different human heavyand light chain acceptor sequences. Based on the alignments with the VHand VL sequences of monoclonal antibody chAb13, the following humansequences were selected as acceptors:

-   -   IGHV4-b*01(0-1) and IGHJ6*01 for constructing heavy chain        acceptor sequences    -   IGKV1-39*01 and IGKJ2*01 for constructing light chain acceptor        sequences

IGHV4-b_IGHJ6 (SEQ ID NO: 176)QVQLQESGPGLVKPSETLSLTCAVSgysissgyywgWIRQPPGKGLEWIGsiyhsgstyynpslksRVTISVDTSKNQFSLKLSSVTAADTAVYYCARxx xxxxxWGQGTTVTVSS;where xxxxxxx represents the CDR-H3 region. IGKV1-39_IGKJ2(SEQ ID NO: 177) DIQMTQSPSSLSASVGDRVTITCrasqsissylnWYQQKPGKAPKLLIYaasslqsGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCxxxxxxxxxFGQ GTKLEIK;where xxxxxxxxx represents the CDR-L3 region.

By grafting the “NW” light chain CDR3 and the remaining fivecorresponding VH and VL CDRs of chAb13 into said acceptor sequences,CDR-grafted, humanized, and modified VH and VL sequences were prepared.To generate humanized antibodies with potential frameworkback-mutations, mutations were identified and introduced into theCDR-grafted antibody sequences by de novo synthesis of the variabledomain, or mutagenic oligonucleotide primers and polymerase chainreactions, or both by methods well known in the art. Differentcombinations of back mutations and other mutations were constructed foreach of the CDR-grafts as follows. Residue numbers for these mutationsare based on the Kabat numbering system.

The following humanized variable regions of the murine monoclonal chAb13antibodies were cloned into IgG expression vectors for functionalcharacterization:

-   -   Humanized Ab13 VH.1 (huAb13VH.1) is a CDR-grafted, humanized        Ab13 VH containing IGHV4-b*01(0-1) and IGHJ6*01 framework        sequences. It also contains a Q1E change to prevent        pyroglutamate formation.    -   Humanized Ab13 VH.1 (huAb13 VH.1a) is a humanized design based        on huAb13VH.1 and contains 9 proposed framework        back-mutation(s): S25T, P40F. K43N, I48M, V671. T68S, V71R,        S79F, R94G.    -   Humanized Ab13 VH.1b (huAb13VH.1b) is an intermediate design        between on huAb13VH.1 and huAb13VH.1a and contains 4 proposed        framework back-mutation(s): K43N, I48M, V671, V71R.    -   Humanized Ab13 VL.1 (huAb13VL.1) is a CDR-grafted, humanized        Ab13 VL containing IGKV139*01 and IGHJ6*01 framework sequences.    -   Humanized Ab13 VL.1a (huAb13VL.1a) is ahumanized design based on        huAb13VL.I and contains 4 proposed framework back-mutation(s):        A43S,L146A, T85E. Y87F.    -   Humanized Ab13 VL.1b (huAb13VL.1b) is anintrmediate dcsign        between on huAb13VL.I and huAb13VL.1a and contains 1 proposed        framework back-mutation(s): Y87F.

The variable region and CDR amino acid sequences of the foregoing aredescribed in Table 18 below.

TABLE 18 Amino acid variable region sequences of humanized Ab13 SEQ IDProtein NO: Clone Region Residues Amino Acid Sequence 143 huAb13VL.1 VLDIQMTQSPSSLSASVGDRVTIT CKASQNVGFNVAWYQQKPGKAP KLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQYNWYPFTFGQGTKLEIK 37 huAb13VL.1 CDR-L1Residues 24-34 KASQNVGFNVA of SEQ ID NO: 143 38 huAb13VL.1 CDR-L2Residues 50-56 SASYRYS of SEQ ID NO: 143 39 huAb13VL.1 CDR-L3Residues 89-97 QQYNWYPFT of SEQ ID NO: 143 144 huAb13VL.1a VLDIQMTQSPSSLSASVGDRVTIT CKASQNVGFNVAWYQQKPGKSP KALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFAEYFC QQYNWYPFTFGQGTKLEIK 37 huAb13VL.1a CDR-L1Residues 24-34 KASQNVGFNVA of SEQ ID NO: 144 38 huAb13VL.1a CDR-L2Residues 50-56 SASYRYS of SEQ ID NO: 144 39 huAb13VL.1a CDR-L3Residues 89-97 QQYNWYPFT of SEQ ID NO: 144 146 huAb13VH.1 VHEVQLQESGPGLVKPSETLSLTC AVSGYSITSGYSWHWIRQPPGK GLEWIGYIHSSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTA ADTAVYYCARYDDYFEYWGQGT TVTVSS 33 huAb13VH.1CDR-H1 Residues 26-36 GYSITSGYSWH of SEQ ID NO: 146 34 huAb13VH.1 CDR-H2Residues 51-66 YIHSSGSTNYNPSLKS of SEQ ID NO: 146 35 huAb13VH.1 CDR-H3Residues 99-105 YDDYFEY of SEQ ID NO: 146 145 huAB13VL.1b VLDIQMTQSPSSLSASVGDRVTIT CKASQNVGFNVAWYQQKPGKAP KLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFC QQYNWYPFTFGQGTKLEIK 37 huAb13VL.1b CDR-L1Residues 24-34 KASQNVGFNVA of SEQ ID NO: 145 38 huAb13VL.1b CDR-L2Residues 50-56 SASYRYS of SEQ ID NO: 145 39 huAb13VL.1b CDR-L3Residues 89-97 QQYNWYPFT of SEQ ID NO: 145 147 huAb13VH.1a VHEVQLQESGPGLVKPSETLSLTC AVTGYSITSGYSWHWIRQFPGN GLEWMGYIHSSGSTNYNPSLKSRISISRDTSKNQFFLKLSSVTA ADTAVYYCAGYDDYFEYWGQGT TVTVSS 33 huAb13VH.1aCDR-H1 Residues 26-36 GYSITSGYSWH of SEQ ID NO: 147 34 huAb13VH.1aCDR-H2 Residues 51-66 YIHSSGSTNYNPSLKS of SEQ ID NO: 147 35 huAb13VH.1aCDR-H3 Residues 99-105 YDDYFEY of SEQ ID NO: 147 148 huAb13VH.1b VHEVQLQESGPGLVKPSETLSLTC AVSGYSITSGYSWHWIRQPPGN GLEWMGYIHSSGSTNYNPSLKSRITISRDTSKNQFSLKLSSVTA ADTAVYYCARYDDYFEYWGQGT TVTVSS 33 huAb13VH.1bCDR-H1 Residues 26-36 GYSITSGYSWH of SEQ ID NO: 148 34 huAb13VH.1bCDR-H2 Residues 51-66 YIHSSGSTNYNPSLKS of SEQ ID NO: 148 35 huAb13VH.1bCDR-H3 Residues 99-105 YDDYFEY of SEQ ID NO: 148

Example 18: Generation of huAb13 Variants

The 3VH and VL region amino acid sequences of humanized Ab3 variantsdescribed in Table 18 were paired together to generate 9huAb13 variantsdescribed in Table 19. The full length amino acid sequences of the heavychain and light chain of the huAb13v1 variant, huAb13v1 are provided inSEQD NOs:168 and 169, respectively.

SEQ ID Protein Amino Acid NO: Clone Region Residues Sequence 147huAb13v1 VH EVQLQESGPGLVKPSETLSLTCAV TGYSITSGYSWHWIRQFPGNGLEWMGYIHSSGSTNYNPSLKSRISISR DTSKNQFFLKLSSVTAADTAVYYC AGYDDYFEYWGQGTTVTVSS33 huAb13vV1 CDR-H1 Residues 26-36 GYSITSGYSWH of SEQ ID NO: 147 34huAb13v1 CDR-H2 Residues 51-66 YIHSSGSTNYNPSLKS of SEQ ID NO: 147 35huAb13v1 CDR-H3 Residues 99-105 YDDYFEY of SEQ ID NO: 147 144 huAb13v1VL DIQMTQSPSSLSASVGDRVTITCK ASQNVGFNVAWYQQKPGKSPKALIYSASYRYSGVPSRFSGSGSGTDFT LTISSLQPEDFAEYFCQQYNWYPF TFGQGTKLEIK 37huAb13v1 CDR-L1 Residues 24-34 KASQNVGFNVA of SEQ ID NO: 144 38 huAb13v1CDR-L2 Residues 50-56 SASYRYS of SEQ ID NO: 144 39 huAb13v1 CDR-L3Residues 89-97 QQYNWYPFT of SEQ ID NO: 144 146 huAb13v2 VHEVQLQESGPGLVKPSETLSLTCAV SGYSITSGYSWHWIRQPPGKGLEWIGYIHSSGSTNYNPSLKSRVTISV DTSKNQFSLKLSSVTAADTAVYYC ARYDDYFEYWGQGTTVTVSS33 huAb13v2 CDR-H1 Residues 26-36 GYSITSGYSWH of SEQ ID NO: 146 34huAb13v2 CDR-H2 Residues 51-66 YIHSSGSTNYNPSLKS of SEQ ID NO: 146 35huAb13v2 CDR-H3 Residues 99-105 YDDYFEY of SEQ ID NO: 146 143 huAb13v2VL DIQMTQSPSSLSASVGDRVTITCK ASQNVGFNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQYNWYPF TFGQGTKLEIK 37huAb13v2 CDR-L1 Residues 24-34 KASQNVGFNVA of SEQ ID NO: 143 38 huAb13v2CDR-L2 Residues 50-56 SASYRYS of SEQ ID NO: 143 39 huAb13v2 CDR-L3Residues 89-97 QQYNWYPFT of SEQ ID NO: 143 146 huAb13v3 VHEVQLQESGPGLVKPSETLSLTCAV SGYSITSGYSWHWIRQPPGKGLEWIGYIHSSGSTNYNPSLKSRVTISV DTSKNQFSLKLSSVTAADTAVYYC ARYDDYFEYWGQGTTVTVSS33 huAb13v3 CDR-H1 Residues 26-36 GYSITSGYSWH of SEQ ID NO: 146 34huAb13v3 CDR-H2 Residues 51-66 YIHSSGSTNYNPSLKS of SEQ ID NO: 146 35huAb13v3 CDR-H3 Residues 99-105 YDDYFEY of SEQ ID NO: 146 144 huAb13v3VL DIQMTQSPSSLSASVGDRVTITCK ASQNVGFNVAWYQQKPGKSPKALIYSASYRYSGVPSRFSGSGSGTDFT LTISSLQPEDFAEYFCQQYNWYPF TFGQGTKLEIK 37huAb13v3 CDR-L1 Residues 24-34 KASQNVGFNVA of SEQ ID NO: 144 38 huAb13v3CDR-L2 Residues 50-56 SASYRYS of SEQ ID NO: 144 39 huAb13v3 CDR-L3Residues 89-97 QQYNWYPFT of SEQ ID NO: 144 146 huAb13v4 VHEVQLQESGPGLVKPSETLSLTCAV SGYSITSGYSWHWIRQPPGKGLEWIGYIHSSGSTNYNPSLKSRVTISV DTSKNQFSLKLSSVTAADTAVYYC ARYDDYFEYWGQGTTVTVSS33 huAb13v4 CDR-H1 Residues 26-36 GYSITSGYSWH of SEQ ID NO: 146 34huAb13v4 CDR-H2 Residues 51-66 YIHSSGSTNYNPSLKS of SEQ ID NO: 146 35huAb13v4 CDR-H3 Residues 99-105 YDDYFEY of SEQ ID NO: 146 145 huAb13v4VL DIQMTQSPSSLSASVGDRVTITCK ASQNVGFNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFT LTISSLQPEDFATYFCQQYNWYPF TFGQGTKLEIK 37huAb13v4 CDR-L1 Residues 24-34 KASQNVGFNVA of SEQ ID NO: 145 38 huAb13v4CDR-L2 Residues 50-56 SASYRYS of SEQ ID NO: 145 39 huAb13v4 CDR-L3Residues 89-97 QQYNWYPFT of SEQ ID NO: 145 147 huAb13v5 VHEVQLQESGPGLVKPSETLSLTCAV TGYSITSGYSWHWIRQFPGNGLEWMGYIHSSGSTNYNPSLKSRISISR DTSKNQFFLKLSSVTAADTAVYYC AGYDDYFEYWGQGTTVTVSS33 huAb13v5 CDR-H1 Residues 26-36 GYSITSGYSWH of SEQ ID NO: 147 34huAb13v5 CDR-H2 Residues 51-66 YIHSSGSTNYNPSLKS of SEQ ID NO: 147 35huAb13v5 CDR-H3 Residues 99-105 YDDYFEY of SEQ ID NO: 147 143 huAb13v5VL DIQMTQSPSSLSASVGDRVTITCK ASQNVGFNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQYNWYPF TFGQGTKLEIK 37huAb13v5 CDR-L1 Residues 24-24 KASQNVGFNVA of SEQ ID NO: 143 38 huAb13v5CDR-L2 Residues 50-56 SASYRYS of SEQ ID NO: 143 39 huAb13v5 CDR-L3Residues 89-97 QQYNWYPFT of SEQ ID NO: 143 147 huAb13v6 VHEVQLQESGPGLVKPSETLSLTCAV TGYSITSGYSWHWIRQFPGNGLEWMGYIHSSGSTNYNPSLKSRISISR DTSKNQFFLKLSSVTAADTAVYYC AGYDDYFEYWGQGTTVTVSS33 huAb13v6 CDR-H1 Residues 26-36 GYSITSGYSWH of SEQ ID NO: 147 34huAb13v6 CDR-H2 Residues 51-66 YIHSSGSTNYNPSLKS of SEQ ID NO: 147 35huAb13v6 CDR-H3 Residues 99-105 YDDYFEY of SEQ ID NO: 147 145 huAb13v6VL DIQMTQSPSSLSASVGDRVTITCK ASQNVGFNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFT LTISSLQPEDFATYFCQQYNWYPF TFGQGTKLSIK 37huAb13v6 CDR-L1 Residues 24-34 KASQNVGFNVA of SEQ ID NO: 145 38 huAb13v6CDR-L2 Residues 50-56 SASYRYS of SEQ ID NO: 145 39 huAb13v6 CDR-L3Residues 89-97 QQYNWYPFT of SEQ ID NO: 145 148 huAb13v7 VHEVQLQESGPGLVKPSETLSLTCAV SGYSITSGYSWHWIRQPPGNGLEWMGYIHSSGSTNYNPSLKSRITISR DTSKNQFSLKLSSVTAADTAVYYC ARYDDYFEYWGQGTTVTVSS33 huAb13v7 CDR-H1 Residues 26-36 GYSITSGYSWH of SEQ ID NO: 148 24huAb13v7 CDR-H2 Residues 51-66 YIHSSGSTNYNPSLKS of SEQ ID NO: 148 35huAb13v7 CDR-H3 Residues 99-105 YDDYFEY of SEQ ID NO: 148 143 huAb13v7VL DIQMTQSPSSLSASVGDRVTITCK ASQNVGFNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQYNWYPF TFGQGTKLEIK 37huAb13v7 CDR-L1 Residues 24-34 KASQNVGFNVA of SEQ ID NO: 143 38 huAb13v7CDR-L2 Residues 50-56 SASYRYS of SEQ ID NO: 143 39 huAb13v7 CDR-L3Residues 89-97 QQYNWYPFT of SEQ ID NO: 143 148 huAb13v8 VHEVQLQESGPGLVKPSETLSLTCAV SGYSITSGYSWHWIRQPPGNGLEWMGYIHSSGSTNYNPSLKSRITISR DTSKNQFSLKLSSVTAADTAVYYC ARYDDYFEYWGQGTTVTVSS33 huAb13v8 CDR-H1 Residues 26-36 GYSITSGYSWH of SEQ ID NO: 148 34huAb13v8 CDR-H2 Residues 51-66 YIHSSGSTNYNPSLKS of SEQ ID NO: 148 35huAb13v8 CDR-H3 Residues 99-105 YDDYFEY of SEQ ID NO: 148 144 huAb13v8VL DIQMTQSPSSLSASVGDRVTITCK ASQNVGFNVAWYQQKPGKSPKALIYSASYRYSGVPSRFSGSGSGTDFT LTISSLQPEDFAEYFCQQYNWYPF TFGQGTKLEIK 37huAb13v8 CDR-L1 Residues 24-34 KASQNVGFNVA of SEQ ID NO: 144 38 huAb13v8CDR-L2 Residues 50-56 SASYRYS of SEQ ID NO: 144 39 huAb13v8 CDR-L3Residues 89-97 QQYNWYPFT of SEQ ID NO: 144 148 huAb13v9 VHEVQLQESGPGLVKPSETLSLTCAV SGYSITSGYSWHWIRQPPGNGLEWMGYIHSSGSTNYNPSLKSRITISR DTSKNQFSLKLSSVTAADTAVYYC ARYDDYFEYWGQGTTVTVSS33 huAb13v9 CDR-H1 Residues 26-36 GYSITSGYSWH of SEQ ID NO: 148 34huAb13v9 CDR-H2 Residues 51-66 YIHSSGSTNYNPSLKS of SEQ ID NO: 148 35huAb13v9 CDR-H3 Residues 99-105 YDDYFEY of SEQ ID NO: 148 145 huAb13v9DIQMTQSPSSLSASVGDRVTITCK ASQNVGFNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFT LTISSLQPEDFATYFCQQYNWYPF TFGQGTKLEIK 37huAb13v9 CDR-L1 Residues 24-34 KASQNVGFNVA of SEQ ID NO: 145 38 huAb13v9CDR-L2 Residues 50-56 SASYRYS of SEQ ID NO: 145 39 huAb13v9 CDR-L3Residues 89-97 QQYNWYPFT of SEQ ID NO: 145

Example 19:Characterization of huAb13 VL.1a Humanized Variants

Nine huAb13 variants described in Examples 17 and 18 were generated andtested for binding to B7-H3 by FACS (according to methods described inExample 6). Six variants did not bind to human B17-1H-3. The remainingthree variants were further characterized for binding by SPR andconjugated (via Method A) to the Bcl-xL inhibitor (specifically thelinker warhead (or synthon) CZ) and assessed for cell cytotoxicity(according to methods described in Example 7). Table 20 provides the invitro characteristics of these variants.

TABLE 20 In vitro characterization of huAb13 VL.1a variants conjugatedto synthon (or linker payload) CZ ELISA Affinity of % hB7- FACS (EC₅₀naked Cytotoxicity DAR agg H3 nM) mAbs (H847 Cell Variant Sequence by byEC₅₀ hB7- cyB7- (Biacore, line IC₅₀) name Number MS SEC nM H3 H3 K_(D))(nM) chAb13-CZ 32, 36 — — 0.26 6.27 18.35 5.7E−09 — huAb13v1- 147, 1444.0 5.1 0.12 6.01 10.0 6.2E−09 0.09 CZ huAb13v5- 147, 143 3.4 2.4 0.195.21 10.59 Poor fit 1.60 CZ huAb13v6- 147, 145 3.6 7.3 0.14 5.83 12.95Poor fit 0.84 CZ

HuAb13v1 was selected for further study due in part to its potent andsuperior cytotoxicity against H847 cells and similar bindingcharacteristics as chAb13 from which it was derived. In contrast,huAb13v and huAb13v6 showed poor fit kinetics in Biacore experimentssuggesting their binding properties are more divergent from the parentalchAb13 than huAb13v1 and have reduced activity in the cell killingassay.

Example 20: In vitro potency of Selected Humanized B7-H3 Antibodies withExemplary Bcl-xL Inhibitor Linker Warheads (Synthons)

Humanized antibodies huAb13v huAb3v2.5 and huAb3v2.6 were selected to beconjugated with several Bcl-xL inhibitor payloads (or synthons) at a 3mg scale using Methods A, E or G, as described in Example 7. Theanti-tumor activity of these ADCs was tested in cytotoxicity assaysusing the NCI-H1650 non-small cell lung cancer cell line as described inExample 7.As control, the in vitro anti-tumor activity of ADCscomprising the non-targeting antibody MSL 109 (a monoclonal antibodythat binds to the CMV glycoprotein H conjugated to Bcl-xL inhibitorpayloads (or synthons) was also evaluated. The results are described inTable 21.

TABLE 21 In vitro tumor cell cytotoxicity of selected humanized B7-H3ADCs with exemplary Bcl-xL inhibitor linker warheads (synthons) % aggADC Conjugation DAR by conc EC₅₀ nM ADC Method by MS SEC (mg/ml) H1650huAb13v1-CZ G 4 3.9 3 0.18 huAb13v1-TX G 3.6 2.8 2.6 0.22 huAb13v1-TV G2.4 3 3.9 0.43 huAb13v1-AAA G 2 20.2 2.7 0.37 huAb13v1-AAD G 3.7 3.3 2.70.21 huAb13v1-WD E 3 5.4 5.8 0.45 huAb13v1-LB A 2.2 21.9 3.7 >133huAb13v1-ZT G 2.4 10.6 1.7 0.3 huAb13v1-ZZ G 1.4 20.3 2.5 0.42huAb13v1-XW G 4.3 6.3 2.6 0.86 huAb13v1-SE A 3.7 4 5.4 0.63 huAb13v1-SRA 2.6 49.5 4.5 0.59 huAb13v1-YG E 3.3 2.1 3.8 0.33 huAb13v1-KZ A 2.816.8 3.5 178.8 huAb3v2.5-CZ G 3.3 15.6 3.6 0.40 huAb3v2.5-TX G 3.3 8.92.9 0.62 huAb3v2.5-TV G 3.7 10.4 3.5 0.53 huAb3v2.5-YY G 2.3 16.2 3.20.71 huAb3v2.5-AAA G 2 14.8 3.3 0.85 huAb3v2.5-AAD G 3.4 11.3 3.7 0.49huAb3v2.5-WD E 2.8 11.5 5.4 0.83 huAb3v2.5-LB A 2.2 24.4 3.9 2.59huAb3v2.5-ZT G 1.6 70.1 3.3 0.95 huAb3v2.5-ZZ G 1.2 19.4 3.7 1.1huAb3v2.5-XW G 3.9 16.4 3.4 2.18 huAb3v2.5-SE A 3.7 10.6 5.4 0.85huAb3v2.5-SR A 1.8 48.5 5.1 0.59 huAb3v2.5-YG E 4 8.6 3.3 0.71huAb3v2.5-KZ A 2.6 24.5 3.4 0.87 huAb3v2.6-CZ G 3.4 15 3.6 0.40huAb3v2.6-TX G 3.2 10.4 3.4 0.47 huAb3v2.6-TV G 3.3 10.7 3.8 0.52huAb3v2.6-YY G 2.2 19.9 3.4 0.72 huAb3v2.6-AAA G 1.9 20.2 3.6 1.24huAb3v2.6-AAD G 3.4 11.9 3.7 0.85 huAb3v2.6-WD E 3.1 12.4 5.3 0.79huAb3v2.6-LB A 2.4 27.2 3.9 2.07 huAb3v2.6-ZT G 1.7 21.6 3.7 1.11huAb3v2.6-ZZ G 1.2 70.7 3.5 1.35 huAb3v2.6-XW G 4 16.8 3.2 2.4huAb3v2.6-SE A 3.6 11.8 5.7 1.01 huAb3v2.6-SR A 2.5 48.2 5.2 0.71huAb3v2.6-YG E 3.7 9.9 4.8 0.68 huAb3v2.6-KZ A 3.5 26.1 3.6 5.52MSL109-CZ G 3.2 0.5 3.7 19.50 MSL109-TX G 3.5 0.7 3 20.00 MSL109-TV G3.6 0 2.6 31.13 MSL109-YY G 2.9 0 1.8 26.53 MSL109-AAA G 1.9 13.7 3.223.52 MSL109-AAD G 3 0.4 3.8 >67 MSL109-WD E 2.9 0 7.06 18.22 MSL109-LBA 1.8 0 4.2 9.88 MSL109-ZT G 2.3 7.5 2.2 >67 MSL109-ZZ G 1.4 15 3.5 >67MSL109-XW G 3.3 3.7 3.2 >67 MSL109-SE A 3.6 33.4 6.0 29.56 MSL109-SR A1.8 2.3 3.8 53.29 MSL109-YG E 3.1 13.2 4.0 19.93 MSL109-KZ A 2.5 18 4.350.16

In contrast to the low anti-tumor activity exhibited by the ADCscomprising the non-targeting antibody MSL109 conjugated to a Bcl-xLinhibitor payload, the B7-H3-targeting ADCs exhibited greater tumor cellkilling, which reflects the antigen-dependent delivery of theB7-H3-targeting ADCs to the B7-H3-expressing tumor cells.

The anti-tumor activity of two of these ADCs was tested in cytotoxicityassays using the NCI-H146 small cell lung cancer cell line as describedin Example 7. The results are described in Table 22.

TABLE 22 In vitro tumor cell cytotoxicity of selected humanized B7-H3ADCs with exemplary Bcl-xL inhibitor synthons ADC Conjugation % agg byconc EC50 nM ADC Method DAR SEC (mg/ml) H146 huAb13v1- 1 2 3.3 11.6 2AAA E2 huAb13v1- 1 2 4.5 14.5 2 WD E2

huAb13v1-AAA E2 and huAb13v1-WD E2 were tested for cytotoxicity usingH146 cells. Both conjugates show potent and comparable cytotoxicity.

Example 21: In vivo analysis of anti-B7-H3 ADCs

Humanized anti-B7-H3 antibodies huAb13v1, huAb3v2.5 and huAb3v2.6 wereselected to be conjugated with several Bcl-xL inhibitor payloads andwere evaluated in xenograf models of small cell lung cancer (H146) asconjugates using a number of Bcl-xL inhibitor warheads (synthons) usingthe methods described in Example 7 and Example 8. The results aresummarized in Table 23 and Table 24.

TABLE 23 In vivo efficacy of humanized anti-B7-H3 ADCs ConjugationDose^([a])/route/ Number TGI_(max) ADC Method DAR regimen of mice (%)TGD (%) AB095 — n/a 6 mg/kg/IP/QDx1 8 0 0 huAb3v2.5-CZ A 3.5 6mg/kg/IP/QDx1 8 92 122 huAb3v2.6-CZ A 3.4 6 mg/kg/IP/QDx1 8 93 130huAb3v2.9-CZ A 2.8 6 mg/kg/IP/QDx1 8 94 135 huAb3v2.9-TX E 1.7 6mg/kg/IP/QDx1 8 93 109 huAb3v2.6-TX E 2.7 6 mg/kg/IP/QDx1 8 92 130huAb3v2.5-TX E 2.5 6 mg/kg/IP/QDx1 8 86 89 ^([a])dose is given inmg/kg/day

TABLE 24 In vivo efficacy of humanized anti-B7-H3 ADCs Conju- Numbergation Dose^([a])/route/ of TGI_(max) ADC Method DAR regimen mice (%)AB095 — n/a 6 mg/kg/IP/QDx1 8 0 huAb3v2.5-AAA E 2.3 6 mg/kg/IP/QDx1 8 65huAb3v2.5-XW E 3.1 6 mg/kg/IP/QDx1 8 51 huAb3v2.6-AAA E 3.5 6mg/kg/IP/QDx1 8 47 huAb3v2.6-XW E 4.0 6 mg/kg/IP/QDx1 8 43 huAb13v1-AAAE 3.5 6 mg/kg/IP/QDx1 8 76 huAb13v1-XW E 4.2 6 mg/kg/IP/QDx1 8 35huAb13v1-TX E2 I 2 6 mg/kg/IP/QDx1 8 88 ^([a])dose is given in mg/kg/day

Humanized anti-B7-H3 antibody huAb13v was conjugated with the Bcl-xLinhibitor synthon WD and evaluated in a xenograft model of theB7-H3-positive small cell lung cancer (H1650) as conjugates using themethods described in Example 7 and Example 8. As control, the in vivoanti-tumor activity of anon-targeting IgG isotype matched antibody(AB095) was also evaluated.

The results are summarized in Table 25.

TABLE 25 In vivo efficacy of humanized anti-B7-H3 ADC huAb13v1-WD inH1650 DAR/ Dose Conjugation mg/kg/ route/ TGI_(max) TGD ADC Method dayregimen (%) (%) AB095^((a)) N.A. 10 IP/QDx1 0 0 huAb13v1- 2/I 1 IP/QDx146* 47* WD-E2 huAb13v1- 2/I 3 IP/QDx1 48* 47* WD-E2 huAb13v1- 2/I 10IP/QDx1 62* 77* WD-E2 ^((a))IgG1 mAb *= p < 0.05 as compared to controltreatment (AB095) ^(¥)= p < 0.05 as compared to the most active partnerin a drug combination N.A. = not applicable

In contrast to the lack of activity observed using the non-targeting IgGisotype-matched antibody Ab095, the B7-H3-targeting Bcl-xL ADCsexhibited tumor growth inhibition (TGI) and tumor growth delay (TGD), asshown in Tables 24 and 25, reflecting the antigen-dependent delivery ofthe B7-H3-targeting ADCs which deliver the Bcl-xL inhibitor to theB7-H3-expressing tumor cells in this xenograft mouse model. As anadditional control, the in vivo anti-tumor activity of ADCs comprisingthe non-targeting antibody MSL109 conjugated with Bcl-xL inhibitorsynthons was evaluated in the xenograft model of the B7-H3-positivesmall cell lung cancer (H1650). The activity of these ADCs was comparedthat of the non-targeting IgG isotype matched antibody, AB095, ascontrol. As shown in Table 26, the ADCs comprising the non-targetingantibody MSL109 conjugated with Bcl-xL inhibitor synthons exhibited verymodest tumor growth inhibition and low or no tumor growth delay. Incontrast, the B7-H3-targeting Bcl-xL ADCs (as shown in Table 25)exhibited, much greater tumor growth inhibition (TGI) and tumor growthdelay (TGD), reflecting the antigen-dependent delivery of these ADCs toB7-H3-expressing cells in this mouse xenograft model.

TABLE 26 In vivo efficacy of non-targeting (MSL109) BCL-xL inhibitingADCs in NCI-H1650 model of NSCLC Growth Inhibition TGI_(max) TGDTreatment Dose^([a])/route/regimen (%) (%) MSL109^(†)-H 3/IP/Q4Dx6 18* 0MSL109^(†)-H 10/IP/Q4Dx6 43* 20* MSL109^(†)-H 30/IP/Q4Dx6 8 0MSL109^(†)-CZ 3/IP/Q4Dx6 29* 0 MSL109^(†)-CZ 3/IP/Q7Dx6 18* 0MSL109^(†)-CZ 10/IP/Q4Dx6 32* 16  MSL109^(†)-CZ 30/IP/Q4Dx6 32* 12 ^(†)Non-targeting antibody ^([a])dose is given in mg/kg/day *= p < 0.05as compared to control treatment (AB095) Q4Dx6 indicates one dose every4 days for a total of 6 doses

Example 22: B7-H3 Combination Therapy

The anti-tumor activity of huAb13v1 as CZ or TX conjugates as purifiedDAR2 (E2) conjugates were characterized in xenograft models of non-smallcell lung cancer (H1650, H1299, H1975, and EBC1) of human origin usingthe methods described in Example 8. The anti-tumor activity was assessedas monotherapy and in combination with docetaxel (H1650, H1299, H1975,and EBC1). The results are presented in Table 27.

TABLE 27 In vivo efficacy of humanized huAb13v1 anti-B7-H3 conjugates asmonotherapy and in combination with docetaxel DAR/ Conjugation Doseroute/ ADC Method mg/kg/day regimen TGI_(max) (%) TGD (%) EBC1 AB095 —10 Q4Dx6/IP  0  0 huAb13v1-TX E2 2/I 10 Q4Dx6/IP 58 67 Docetaxel — 7.5QDx1/IV 85 80 huAb13v1-TX E2 + 2/I 10 + 7.5 Q4Dx6/IP + Q 140  140 Docetaxel Dx1/IV NCI-H1299 AB095 10 Q4Dx6/IP  0  0 huAb13v1-TX E2 2/I 10Q4Dx6/IP 80 24 Docetaxel — 7.5 QDx1/IV 87 48 huAb13v1-TX E2 + 2/I 10 +7.5 Q4Dx6/IP + Q 97 83 Docetaxel Dx1/IV NCI-H1975 AB095 10 Q4Dx6/IP  0 0 huAb13v1-TX E2 2/I 10 Q4Dx6/IP 52 62 Docetaxel 7.5 QDx1/IV 81 77huAb13v1-TX E2 + 2/I 10 + 7.5 Q4Dx6/IP + Q 92 108  Docetaxel Dx1/IVNCI-H1650 AB095 — 8 Q7Dx6/IP  0  0 huAb13v1-CZ 2/I 10 QDx1/IP 80 100 Docetaxel — 7.5 QDx1/IV 84 143  huAb13v1-CZ + Docetaxel — 10 + 7.5QDx1/IP + Q 99 >600  Dx1/IV AB095^((a)) N.A. 10 IP/Q14Dx3  0  0 DTX N.A.7.5 IV/Q14Dx3  80* 158* huAb13v1-WD E2 2/I 10 IP/Q14Dx3  67*  83*huAb13v1-WD E2 + 2/I + 10 + 7.5 IP/Q14Dx3 +   98*^(¥) >717*^(¥ ) DTX)N.A. IV/Q14Dx3 huAb13v1-WD E2 2/. 3 IP/Q14Dx3  56*  75* huAb13v1-WD E2 +2/I +  3 + 7.5 IP/Q14Dx3 +   99*^(¥) >717*^(¥ ) DTX) N.A. IV/Q14Dx3huAb13v1-WD E2 2/I 1 IP/Q14Dx3  60*  67* huAb13v1-WD E2 + 2/I +  1 + 7.5IP/Q14Dx3 +   88*^(¥)  467*^(¥) DTX N.A IV/Q14Dx3 huAb13v1-AAA E2 2/I 10IP/Q14Dx3  63* 117* huAb13v1-AAA E2 + 2/I + 10 + 7.5 IP/Q14Dx3 +  99*^(¥) >717*^(¥ ) DTX N.A IV/Q14Dx3 huAb13v1-AAA E2 2/I 3 IP/Q14Dx3 60* 117* huAb13v1-AAA E2 + 2/I +  3 + 7.5 IP/Q14Dx3 +   99*^(¥)>717*^(¥ ) DTX N.A IV/Q14Dx3 huAb13v1-AAA E2 2/I 1 IP/Q14Dx3  50*  67*huAb13v1-AAA E2 + 2/I +  1 + 7.5 IP/Q14Dx3 +   92*^(¥) >717*^(¥ ) DTXN.A IV/Q14Dx3 ^((a))IgG1 mAb *= p < 0.05 as compared to controltreatment (AB095) ^(¥)= p < 0.05 as compared to the most active partnerin a drug combination N.A. = not applicable

The results presented in Table 27 demonstrate that above, huAb13v1 asCZ, TX, WD or AAA purified DAR2 (E2) conjugates inhibited the growth ofall four NSCLC xenograft models as monotherapy. In addition, huAb13v1 asCZ, TX, WD or AAA purified DAR2 (E2) conjugates effectively combinedwith docetaxel to produce more sustained tumor growth inhibition. Thisis most dramatically illustrated in the H1650 xenograft model where thecombination therapy resulted in a TGD of between 467% and >717%, whereasthe individual monotherapies resulted in TGD in the range of 67%-158%.These results support the clinical utility of Bcl-xL inhibitor (Bcl-xLi)ADCs to be dosed in combination with chemotherapy.

SEQUENCE SUMMARY SEQ ID NO: Description Amino Acid Sequence 1chAb2 VH amino acid sequence QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWI GMIHPDSGTTNYNEKFRSKATLTVDKSSSTAYMQLSSLTSEDSAVYYC AVYYGSTYWYFDVWGTGTTVTVSS 2chAb2 VH CDR1 amino acid sequence GYTFTSYWMH 3chAb2 VH CDR2 amino acid sequence MIHPDSGTTNYNEKFRS 4chAb2 VH CDR3 amino acid sequence YYGSTYWYFDV 5chAb2 VL amino acid sequence DVVMTQTPLSLPVSLGDQAYISCRSSQSLVHINGNTYLHWYRQKPGQS PKLLIYKVSNRFSGVPDRFSGSGSGIDFILKISRVEAEDLGVYFCSQS THFPFTFGSGTKLEIK 6chAb2 VL CDR1 amino acid sequence RSSQSLVHINGNTYLH 7chAb2, chAb3, chAb10, huAb3VL.1, KVSNRFShuAb3VL.1a, huAb3VL.1b, huAb3v2.1, huAb3v2.2, huAb3v2.3, huAb3v2.4,huAb3v2.5, huAb3v2.6, huAb3v2.7, huAb3v2.8, and huAb3v2.9 VL CDR2 aminoacid sequence 8 chAb2 VL CDR3 amino acid sequence SQSTHFPFT 9chAb3 VH amino acid sequence QVQLQQPGAELVKPGASVKLSCKASGYTFSSYWMHWVKQRPGQGLEWI GLIHPDSGSTNYNEMFKNKATLTVDRSSSTAYVQLSSLTSEDSAVYFC AGGGRLYFDYWGQGTTLIVSS 10chAb3, huAb3VH.1, huAb3VH.1a, GYTFSSYWMHhuAb3VH.1b, huAb3v2.1, huAb3v2.2, huAb3v2.3, huAb3v2.4, huAb3v2.5,huAb3v2.6, huAb3v2.7, huAb3v2.8, andhuAb3v2.9 VH CDR1 amino acid sequence 11chAb3, huAb3VH.1, huAb3VH.1a, and LIHPDSGSTNYNEMFKNhuAb3VH.1b VH CDR2 amino acid sequence 12 chAb3, huAb3VH.1, huAb3VH.1a,GGRLYFDY huAb3VH.1b, huAb3v2.1, huAb3v2.2,huAb3v2.3, huAb3v2.4, huAb3v2.5, huAb3v2.6, huAb3v2.7, huAb3v2.8, andhuAb3v2.9 VH CDR3 amino acid sequence 13 chAb3 VL amino acid sequenceDVVMTQTPLSLPVSLGDQASISCR SSQSLVHSNGDTYLRWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGS GTDFTLKITRVEAEDLGVYFCSQS THVPYTFGGGTKLEIK 14chAb3, huAb3VL.1, huAb3VL.1a, and RSSQSLVHSNGDTYLRhuAb3VL.1b VL CDR1 amino acid sequence 15 chAb3, huAb3VL.1, huAb3VL.1a,SQSTHVPYT huAb3VL.1b, huAb3v2.1, huAb3v2.2,huAb3v2.3, huAb3v2.4, huAb3v2.5, huAb3v2.6, huAb3v2.7, huAb3v2.8, andhuAb3v2.9 VL CDR3 amino acid sequence 16 chAb4 VH amino acid sequenceQVQLQQPGAELVKPGASVKLSCKA SGYSFTSYWMHWVKQRPGQGLEWIGMIHPNSGSNNYNEKFKSKATLTV DKSSNTAYMQLSSLTSEDSAVYYC ARRLGLHFDYWGQGTTLIVSS17 chAb4 VH CDR1 amino acid sequence GYSFTSYWMH 18chAb4 VH CDR2 amino acid sequence MIHPNSGSNNYNEKFKS 19chAb4 VH CDR3 amino acid sequence RLGLHFDY 20chAb4 VL amino acid sequence DIVMTQSQKFMSTPVGDRVSITCKASQNVGTAVAWYQQKPGQSPKLLI YSASNRYTGVPDRFTGSGSGTDFTLTISNMQSEDLADYFCQQYSSYPY TFGGGTKLEIK 21chAb4 VL CDR1 amino acid sequence KASQNVGTAVA 22chAb4 VL CDR2 amino acid sequence SASNRYT 23chAb4 VL CDR3 amino acid sequence QQYSSYPYT 24chAb18 VH amino acid sequence QVQLQQSAAELARPGASVKMSCKASGYSFTSYTIHWVKQRPGQGLEWI GYINPNSRNTDYNQKFKDETTLTADRSSSTAYMQLISLTSEDSAVYYC ARYSGSTPYWYFDVWGAGTTVTVS S 25chAb18, huAb18VH.1, huAb18VH.1a, and GYSFTSYTIHhuAb18VH.1b VH CDR1 amino acid sequence 26chAb18, huAb18VH.1, and huAb18VH.1a VH YINPNSRNTDYNQKFKDCDR2 amino acid sequence 27 chAb18, huAb18VH.1, huAb18VH.1a, andYSGSTPYWYFDV huAb18VH.1b VH CDR3 amino acid sequence 28chAb18 VL amino acid sequence QIVLTQSPAILSASPGEKVTMTCRASSSVSYMNWYQQKPGSSPKPWIY ATSNLASGVPARFSVSVSGTSHSLTISRVEAEDAATYYCQQWSSNPLT FGAGTKLELK 29 chAb18, huAb18VL.1, huAb18VL.1a,RASSSVSYMN huAb18VL.1b, huAb18VL.2, and huAb18VL.2a, VL CDR1 amino acidsequence 30 chAb18, huAb18VL.1, huAb18VL.1a, ATSNLAShuAb18VL.1b, huAb18VL.2, and huAb18VL.2a, VL CDR2 amino acid sequence 31chAb18, huAb18VL.1, huAb18VL.1a, QQWSSNPLT huAb18VL.1b, huAb18VL.2, andhuAb18VL.2a, VL CDR3 amino acid sequence 32chAb13 VH amino acid sequence DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYSWHWIRQFPGNKLEW MGYIHSSGSTNYNPSLKSRISINRDTSKNQFFLQLNSVTTEDTATYYC AGYDDYFEYWGQGTTLTVSS 33chAb13, huAb13Vh.1, huAb13Vh.1a, GYSITSGYSWHhuAb13Vh.1b, huAb13v1, huAb13v2, huAb13v3, huAb13v4, huAb13v5, huAb13v6,huAb13v7, huAb13v8, and huAb13v9 VH CDR1 amino acid sequence 34chAb13, huAb13Vh.1, huAb13Vh.1a, YIHSSGSTNYNPSLKShuAb13Vh.1b, huAb13v1, huAb13v2, huAb13v3, huAb13v4, huAb13v5, huAb13v6,huAb13v7, huAb13v8, and huAb13v9 VH CDR2 amino acid sequence 35chAb13, huAb13Vh.1, huAb13Vh.1a, YDDYFEYhuAb13Vh.1b, huAb13v1, huAb13v2, huAb13v3, huAb13v4, huAb13v5, huAb13v6,huAb13v7, huAb13v8, and huAb13v9 VH CDR3 amino acid sequence 36chAb13 VL amino acid sequence DIVMTQSQKFMSTSVGDRVSVTCKASQNVGFNVAWYQQKPGQSPKALI YSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYPF TFGSGTKLEIK 37 chAb13, huAb13VL.1, huAb13VL.1a,KASQNVGFNVA huAb13VL.1b, huAb13v1, huAb13v2,huAb13v3, huAb13v4, huAb13v5, huAb13v6,huAb13v7, huAb13v8, and huAb13v9 VL CDR1 amino acid sequence 38chAb13, huAb13VL.1, huAb13VL.1a, SASYRYShuAb13VL.1b, huAb13v1, huAb13v2, huAb13v3, huAb13v4, huAb13v5, huAb13v6,huAb13v7, huAb13v8, and huAb13v9 VL CDR2 amino acid sequence 39huAb13VL.1, huAb13VL.1a, huAb13VL.1b, QQYNWYPFThuAb13v1, huAb13v2, huAb13v3, huAb13v4,huAb13v5, huAb13v6, huAb13v7, huAb13v8, and huAb13v9 VL CDR3 amino acidsequence 40 chAb12 VH amino acid sequence EVQLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEKRLEWV ATISSGTNYTYYPDSVKGRFTISRDNAKNTLYLQMTSLRSEDTAMYYC ARQGRYSWIAYWGQGTLVTVSA 41chAb12 VH CDR1 amino acid sequence GFTFSSYAMS 42chAb12 VH CDR2 amino acid sequence TISSGTNYTYYPDSVKG 43chAb12 VH CDR3 amino acid sequence QGRYSWIAY 44chAb12 VL amino acid sequence DIVLTQSPASLAVSLGQRATISCRASKSVSTSDYSYMHWNQQKPGQPP KLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSR ELLTFGAGTKLELK 45chAb12 VL CDR1 amino acid sequence RASKSVSTSDYSYMH 46chAb12and chAb17 VL CDR2 amino acid LASNLES 47chAb12 VL CDR3 amino acid sequence QHSRELLT 48chAb14 VH amino acid sequence EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPEKRLEWV ATISGGGTNTYYPDSVEGRFTISRDNAKNFLYLQMSSLRSEDTALYYC ARHYGSQTMDYWGQGTSVTVSS 49chAb14 and chAb8 VH CDR1 amino acid GFTFSSYGMS sequence 50chAb14 VH CDR2 amino acid sequence TISGGGTNTYYPDSVEG 51chAb14 VH CDR3 amino acid sequence HYGSQTMDY 52chAb14 VL amino acid sequence DIQMTQSPASLSASVGETVTITCRTSGNIHNYLTWYQQKQGKSPQLLV YNAKTLADGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQHFWSIMW TFGGGTKLEIK 53chAb14 VL CDR1 amino acid sequence RTSGNIHNYLT 54chAb14 VL CDR2 amino acid sequence NAKTLAD 55chAb14 VL CDR3 amino acid sequence QHFWSIMWT 56chAb6 VH amino acid sequence QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWI GEILPGSGSTNYNEKFKGKATFTADTSSNTAYMQVSSLTSEDSAVHYC ARRGYGYVPYALDYWGQGTSVIVS S 57chAb6 VH CDR1 amino acid sequence GYTFSRYWIE 58chAb6 VH CDR2 amino acid sequence EILPGSGSTNYNEKFKG 59chAb6 VH CDR3 amino acid sequence RGYGYVPYALDY 60chAb6 VL amino acid sequence EIQMTQTTSSLSASLGDRVTISCRASQDISNSLNWYQQKPDGTVNLLI YYTSRLYSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPY TFGGGTKLEIK 61chAb6 VL CDR1 amino acid sequence RASQDISNSLN 62chAb6 VL CDR2 amino acid sequence YTSRLYS 63chAb6 VL CDR3 amino acid sequence QQGNTLPYT 64chAb11 VH amino acid sequence EVKLVESGGGLVQPGGSLRLSCATSGFTFTNYYMSWVRQPPGKALEWL GFIRNKANDYTTEYSASVKGRFTISRDNSQSILYLQMNTLRAEDSATY YCARESPGNPFAYWGQGTLVTVSA 65chAb11 VH CDR1 amino acid sequence GFTFTNYYMS 66chAb11 VH CDR2 amino acid sequence FIRNKANDYTTEYSASVKG 67chAb11 VH CDR3 amino acid sequence ESPGNPFAY 68chAb11 VL amino acid sequence DIVMTQSPSSLTVTAGEKVTMTCKSSQSLLNSGTQKNFLTWYQQKPGQ PPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYFCQN DYIYPLTFGAGTKLELK 69chAb11 VL CDR1 amino acid sequence KSSQSLLNSGTQKNFLT 70chAb11 VL CDR2 amino acid sequence WASTRES 71chAb11 VL CDR3 amino acid sequence QNDYIYPLT 72chAb16 VH amino acid sequence EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWI GEINPDSSTINYTPSLKDKFIISRDNAKNTLYLQMSKVRSEDTALYYC ARPGFGNYIYAMDYWGQGTSVTVS S 73chAb16 VH CDR1 amino acid sequence GFDFSRYWMS 74chAb16 VH CDR2 amino acid sequence EINPDSSTINYTPSLKD 75chAb16 VH CDR3 amino acid sequence PGFGNYIYAMDY 76chAb16 VL amino acid sequence DIQMTQTTSSLSASLGDRVTINCRASQDISNFLNWYQQKPDGTVKLLI YYTSRLYLGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPP TFGGGTKLEIK 77chAb16 VL CDR1 amino acid sequence RASQDISNFLN 78chAb16 VL CDR2 amino acid sequence YTSRLYL 79chAb16 VL CDR3 amino acid sequence QQGNTLPPT 80chAb10 VH amino acid sequence DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNRLEW MGHINYSGITNYNPSLKSRISITRDTSKNQFFLQLYSVTTEDTATYFC ARRSLFYYYGSSLYAMDYWGQGTS VTVSS 81chAb10 VH CDR1 amino acid sequence GYSITSDYAWN 82chAb10 VH CDR2 amino acid sequence HINYSGITNYNPSLKS 83chAb10 VH CDR3 amino acid sequence RSLFYYYGSSLYAMDY 84chAb10 VL amino acid sequence DVVMTQSPFSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQS PKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQS THVPWTFGGGTKLEIK 85chAb10 VL CDR1 amino acid sequence RSSQSLVHSNGNTYLH 86chAb10 VL CDR3 amino acid sequence SQSTHVPWT 87chAb7 VH amino acid sequence EVQLVESGENLVKPGGSLKLSCAASGFSFRGYGMSWVRQTPDKRLEWV AAISTGGNYTYYPDSVQGRFTISRDNANNTLYLQMSSLKSEDTAMYYC ARRGGNYAGFAYWGQGTLVTVSA 88chAb7 VH CDR1 amino acid sequence GFSFRGYGMS 89chAb7 VH CDR2 amino acid sequence AISTGGNYTYYPDSVQG 90chAb7 VH CDR3 amino acid sequence RGGNYAGFAY 91chAb7 VL amino acid sequence DIQMTQSPASLSVSVGETVTITCRPSENIYSNLAWYQQKQGKSPQLLV YAATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHFWGTPF TFGSGTKLEIK 92chAb7 VL CDR1 amino acid sequence RPSENIYSNLA 93chAb7 and chAb8 VL CDR2 amino acid AATNLAD 94chAb7 VL CDR3 amino acid sequence QHFWGTPFT 95chAb8 VH amino acid sequence EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPEKRLEWV ATISGGGNYTYCPDSVKGRFTISRDNAKNNLYLQMSSLRSEDTALYYC TRQRGYDYHYAMDFWGQGTSVIVS S 96chAb8 VH CDR2 amino acid sequence TISGGGNYTYCPDSVKG 97chAb8 VH CDR3 amino acid sequence QRGYDYHYAMDF 98chAb8 VL amino acid sequence DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWHQQKQGKSPQLLV YAATNLADGVPSRFSGNGSDTQYSLKINSLQSEDFGSYFCQNFWGTSW TFGGGTKLEIK 99chAb8 VL CDR1 amino acid sequence RASENIYSNLA 100chAb8 VL CDR3 amino acid sequence QNFWGTSWT 101chAb17 VH amino acid sequence EVKLVESGGGLVQPGGSLKLSCAASGFTFSSYIMSWVRQTPEKRLEWV ASIVSSNITYYPDSMKGRFTISRDNARNILYLQMSSLKSEDTAMYYCA RSGTRAWFAYWGQGTLVTVSA 102chAb17 VH CDR1 amino acid sequence GFTFSSYIMS 103chAb17 VH CDR2 amino acid sequence SIVSSNITYYPDSMKG 104chAb17 VH CDR3 amino acid sequence SGTRAWFAY 105chAb17 VL amino acid sequence DIVLTQSPASLAVSLGQRATISCRASKSVSTSAYSYMHWYQQKPGQPP KLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSR ELPYTFGGGTKLEIK 106chAb17 VL CDR1 amino acid sequence RASKSVSTSAYSYMH 107chAb17 VL CDR3 amino acid sequence QHSRELPYT 108chAb5 VH amino acid sequence QVQLQQPGDELVKPGASVKLSCKTSGYTFTTDWMHWVKQRPGQGLEWI GMIHPNSGTTNYNEKFKSKAALTVDKSSSTACMQLSSLTSEDSAVYYC ARSYWKWYFDVWGIGTTVIVSS 109chAb5 VH CDR1 amino acid sequence GYTFTTDWMH 110chAb5 VH CDR2 amino acid sequence MIHPNSGTTNYNEKFKS 111chAb5 VH CDR3 amino acid sequence SYWKWYFDV 112chAb5 VL amino acid sequence QIVLTQSPAIMSASLGEEITLTCSASSSVSYMHWYQQKSGTSPKLLIY STSNLASGVPSRFSGSGSGTFYSLTISSVEAEDSADYYCHQWTSYMYT FGGGTKLEIK 113chAb5 VL CDR1 amino acid sequence SASSSVSYMH 114chAb5 VL CDR2 amino acid sequence STSNLAS 115chAb5 VL CDR3 amino acid sequence HQWTSYMYT 116huAb18VH.1, huAbl8v1, and huAb18v5 VH EVQLVQSGAEVKKPGSSVKVSCKAamino acid sequence SGYSFTSYTIHWVRQAPGQGLEWM GYINPNSRNTDYNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYC ARYSGSTPYWYFDVWGQGTIVTVS S 117huAb18VH.1a, huAb18v3, huAb18v8, and EVQLVQSGAEVKKPGSSVKVSCKAhuAb18v9 VH amino acid sequence SGYSFTSYTIHWVRQAPGQGLEWIGYINPNSRNTDYNQKFKDRTTLTA DRSTSTAYMELSSLRSEDTAVYYCARYSGSTPYWYFDVWGQGTIVTVS S 118 huAb18VH.1b, huAb18v2, huAb18v4,EVQLVQSGAEVKKPGSSVKVSCKA huAb18v6, huAb18v7, and huAb18v10 VHSGYSFTSYTIHWVRQAPGQGLEWM amino acid sequence GYINPNSRNTDYAQKFQGRVTLTADKSTSTAYMELSSLRSEDTAVYYC ARYSGSTPYWYFDVWGQGTIVTVS S 119huAb18VH.1b VH CDR2 amino acid sequence YINPNSRNTDYAQKFQG 120huAb18VL.1, huAbl8v1, and huAb18v2 VL DIQLTQSPSFLSASVGDRVTITCRamino acid sequence ASSSVSYMNWYQQKPGKAPKLLIY ATSNLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQWSSNPLT FGQGTKLEIK 121huAb18VL.1a, huAb18v3, and huAb18v4 VL DIQLTQSPSFLSASVGDRVTITCRamino acid sequence ASSSVSYMNWYQQKPGKSPKPWIY ATSNLASGVPSRFSVSVSGTEHTLTISSLQPEDFATYYCQQWSSNPLT FGQGTKLEIK 122huAb18VL.1b, huAb18v8, and huAb18v10 VL DIQLTQSPSFLSASVGDRVTITCRamino acid sequence ASSSVSYMNWYQQKPGKAPKPWIY ATSNLASGVPSRFSVSGSGTEHTLTISSLQPEDFATYYCQQWSSNPLT FGQGTKLEIK 123huAb18VL.2, huAb18v5, and huAb18v6 VL EIVLTQSPDFQSVTPKEKVTITCRamino acid sequence ASSSVSYMNWYQQKPDQSPKLLIK ATSNLASGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQWSSNPLT FGQGTKLEIK 124huAb18VL.2a, huAb18v7, and huAb18v9 VL EIVLTQSPDFQSVTPKEKVTITCRamino acid sequence ASSSVSYMNWYQQKPDQSPKPWIY ATSNLASGVPSRFSVSVSGTDHTLTINSLEAEDAATYYCQQWSSNPLT FGQGTKLEIK 125huAb3VH.1, huAb3v1, and huAb3v4 VH EVQLVQSGAEVKKPGSSVKVSCKAamino acid sequence SGYTFSSYWMHWVRQAPGQGLEWM GLIHPDSGSTNYNEMFKNRVTITADKSTSTAYMELSSLRSEDTAVYYC ARGGRLYFDYWGQGTTVIVSS 126huAb3VH.1a, huAb3v3, and huAb3v6 VH EVQLVQSGAEVKKPGSSVKVSCKAamino acid sequence SGYTFSSYWMHWVRQAPGQGLEWI GLIHPDSGSTNYNEMFKNRATLTVDRSTSTAYVELSSLRSEDTAVYFC AGGGRLYFDYWGQGTTVIVSS 127huAb3VH.1b, huAb3v2, and huAb3v5 VH EVQLVQSGAEVKKPGSSVKVSCKAamino acid sequence SGYTFSSYWMHWVRQAPGQGLEWI GLIHPDSGSTNYNEMFKNRATLTVDRSTSTAYMELSSLRSEDTAVYYC AGGGRLYFDYWGQGTTVIVSS 128huAb3VL.1, huAb3v1, and huAb3v2 VL DIVMTQSPLSLPVTPGEPASISCRamino acid sequence SSQSLVHSNGDTYLRWYLQKPGQS PQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQS THVPYTFGGGTKVEIK 129huAb3VL.1a and huAb3v3 VL amino acid DVVMTQSPLSLPVTPGEPASISCR sequenceSSQSLVHSNGDTYLRWYLQKPGQS PQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQS THVPYTFGGGTKVEIK 130huAb3VL.1b, huAb3v4, huAb3v5, and DVVMTQSPLSLPVTPGEPASISCRhuAb3v6 VL amino acid sequence SSQSLVHSNGDTYLRWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGS GTDFTLKISRVEAEDVGVYYCSQS THVPYTFGGGTKVEIK 131huAb3v2.1, huAb3v2.2, and huAb3v2.3 VH EVQLVQSGAEVKKPGSSVKVSCKAamino acid sequence SGYTFSSYWMHWVRQAPGQGLEWI GLIHPWSGSTNYNEMFKNRATLTVDRSTSTAYMELSSLRSEDTAVYYC AGGGRLYFDYWGQGTTVIVSS 132huAb3v2.1, huAb3v2.2, and huAb3v2.3 VH LIHPWSGSTNYNEMFKNCDR2 amino acid sequence 133 huAb3v2.1, huAb3v2.4, and huAb3v2.7 VLDIVMTQSPLSLPVTPGEPASISCR amino acid sequence SSQSLVHSSGDTYLRWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGS GTDFTLKISRVEAEDVGVYYCSQS THVPYTFGGGTKVEIK 134huAb3v2.1, huAb3v2.4, and huAb3v2.7 VL RSSQSLVHSSGDTYLRCDR1 amino acid sequence 135 huAb3v2.2, huAb3v2.5, and huAb3v2.8 VLDIVMTQSPLSLPVTPGEPASISCR amino acid sequence SSQSLVHSNRDTYLRWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGS GTDFTLKISRVEAEDVGVYYCSQS THVPYTFGGGTKVEIK 136huAb3v2.2, huAb3v2.5, and huAb3v2.8 VL RSSQSLVHSNRDTYLRCDR1 amino acid sequence 137 huAb3v2.3, huAb3v2.6, and huAb3v2.9 VLDIVMTQSPLSLPVTPGEPASISCR amino acid sequence SSQSLVHSNQDTYLRWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGS GTDFTLKISRVEAEDVGVYYCSQS THVPYTFGGGTKVEIK 138huAb3v2.3, huAb3v2.6, and huAb3v2.9 VL RSSQSLVHSNQDTYLRCDR1 amino acid sequence 139 huAb3v2.4, huAb3v2.5, and huAb3v2.6 VHEVQLVQSGAEVKKPGSSVKVSCKA amino acid sequence SGYTFSSYWMHWVRQAPGQGLEWIGLIHPESGSTNYNEMFKNRATLTV DRSTSTAYMELSSLRSEDTAVYYC AGGGRLYFDYWGQGTTVIVSS140 huAb3v2.4, huAb3v2.5, and huAb3v2.6 LIHPESGSTNYNEMFKNVH CDR2 amino acid sequence 141 huAb3v2.7, huAb3v2.8, and huAb3v2.9 VHEVQLVQSGAEVKKPGSSVKVSCKA amino acid sequence SGYTFSSYWMHWVRQAPGQGLEWIGLIHPISGSTNYNEMFKNRATLTV DRSTSTAYMELSSLRSEDTAVYYC AGGGRLYFDYWGQGTTVIVSS142 huAb3v2.7, huAb3v2.8, and huAb3v2.9 VH LIHPISGSTNYNEMFKNCDR2 amino acid sequence 143 huAb13VL.1, huAb13v2, huAb13v5, andDIQMTQSPSSLSASVGDRVTITCK huAb13v7 VL amino acid sequenceASQNVGFNVAWYQQKPGKAPKLLI YSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNWYPF TFGQGTKLEIK 144huAb13VL.1a, huAb13v1, huAb13v3, and DIQMTQSPSSLSASVGDRVTITCKhuAb13v8 VL amino acid sequence ASQNVGFNVAWYQQKPGKSPKALIYSASYRYSGVPSRFSGSGSGTDFT LTISSLQPEDFAEYFCQQYNWYPF TFGQGTKLEIK 145huAB13VL.1b, huAb13v4, huAb13v6, and DIQMTQSPSSLSASVGDRVTITCKhuAb13v9 VL amino acid sequence ASQNVGFNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFT LTISSLQPEDFATYFCQQYNWYPF TFGQGTKLEIK 146huAb13VH.1, huAb13v2, huAb13v3, and EVQLQESGPGLVKPSETLSLTCAVhuAb13v4 VH amino acid sequence SGYSITSGYSWHWIRQPPGKGLEWIGYIHSSGSTNYNPSLKSRVTISV DTSKNQFSLKLSSVTAADTAVYYC ARYDDYFEYWGQGTTVTVSS147 huAb13VH.1a, huAb13v1, huAb13v5, and EVQLQESGPGLVKPSETLSLTCAVhuAb13v6 VH amino acid sequence TGYSITSGYSWHWIRQFPGNGLEWMGYIHSSGSTNYNPSLKSRISISR DTSKNQFFLKLSSVTAADTAVYYC AGYDDYFEYWGQGTTVTVSS148 huAb13VH.1b, huAb13v7, huAb13v8, and EVQLQESGPGLVKPSETLSLTCAVhuAb13v9 VH amino acid sequence SGYSITSGYSWHWIRQPPGNGLEWMGYIHSSGSTNYNPSLKSRITISR DTSKNQFSLKLSSVTAADTAVYYC ARYDDYFEYWGQGTTVTVSS149 B7-H3 amino acid sequence (human) MLRRRGSPGMGVHVGAALGALWFCLTGALEVQVPEDPVVALVGTDATL CCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALF PDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSK PSMTLEPNKDLRPGDTVTITCSSYQGYPEAEVFWQDGQGVPLIGNVIT SQMANEQGLFDVHSILRVVLGANGTYSCLVRNPVLQQDAHSSVTITPQ RSPTGAVEVQVPEDPVVALVGTDATLRCSFSPEPGFSLAQLNLIWQLT DTKQLVHSFTEGRDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEG SFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCS SYRGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSVLRVVLGA NGTYSCLVRNPVLQQDAHGSVTITGQPMTFPPEALWVTVGLSVCLIAL LVALAFVCWRKIKQSCEEENAGAEDQDGEGEGSKTALQPLKHSDSKED DGQEIA 150 Human B7-H3-ECD (fc fusion)MLRRRGSPGMGVHVGAALGALWFC Note: LTGALEVQVPEDPVVALVGTDATLFc sequence is underlined CCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALF PDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSK PSMTLEPNKDLRPGDTVTITCSSYQGYPEAEVFWQDGQGVPLTGNVTT SQMANEQGLFDVHSILRVVLGANGTYSCLVRNPVLQQDAHSSVTITPQ RSPTGAVEVQVPEDPVVALVGTDATLRCSFSPEPGFSLAQLNLIWQLT DTKQLVHSFTEGRDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEG SFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCS SYRGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSVLRVVLGA NGTYSCLVRNPVLQQDAHGSVTITGQPMTFAAADKTHTCPPCPAPEAE GAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 151Mouse B7-H3-ECD (fc fusion) MLRGWGGPSVGVCVRTALGVLCLC Note:LTGAVEVQVSEDPVVALVDTDATL Fc sequence is underlinedRCSFSPEPGFSLAQLNLIWQLTDT KQLVHSFTEGRDQGSAYSNRTALFPDLLVQGNASLRLQRVRVTDEGSY TCFVSIQDFDSAAVSLQVAAPYSKPSMTLEPNKDLRPGNMVTITCSSY QGYPEAEVFWKDGQGVPLTGNVTTSQMANERGLFDVHSVLRVVLGANG TYSCLVRNPVLQQDAHGSVTITGQPLTFAAADKTHTCPPCPAPEAEGA PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 152Human B7-H3-ECD (his tag) MEFGLSWLFLVAILKGVQCGALEVQVPEDPVVALVGTDATLCCSFSPE PGFSLAQLNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQG NASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEP NKDLRPGDTVTITCSSYQGYPEAEVFWQDGQGVPLTGNVTTSQMANEQ GLFDVHSILRVVLGANGTYSCLVRNPVLQQDAHSSVTITPQRSPTGAV EVQVPEDPVVALVGTDATLRCSFSPEPGFSLAQLNLIWQLTDTKQLVH SFTEGRDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVS IRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYRGYPE AEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSVLRVVLGANGTYSCL VRNPVLQQDAHGSVTITGQPMTHH HHHH 153Mouse B7-H3-ECD (his tag) MEFGLSWLFLVAILKGVQCVEVQVSEDPVVALVDTDATLRCSFSPEPG FSLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYSNRTALFPDLLVQGNA SLRLQRVRVTDEGSYTCFVSIQDFDSAAVSLQVAAPYSKPSMTLEPNK DLRPGNMVTITCSSYQGYPEAEVFWKDGQGVPLTGNVTTSQMANERGL FDVHSVLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQPLTFHHHHH H 154 Cynomolgus B7-H3-ECD (his tag)MLHRRGSPGMGVHVGAALGALWFC LTGALEVQVPEDPVVALVGTDATLRCSFSPEPGFSLAQLNLIWQLTDT KQLVHSFTEGRDQGSAYANRTALFLDLLAQGNASLRLQRVRVADEGSF TCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSY RGYPEAEVFWQDGQGAPLTGNVTTSQMANEQGLFDVHSVLRVVLGANG TYSCLVRNPVLQQDAHGSITITPQRSPTGAVEVQVPEDPVVALVGTDA TLRCSF SPEPGFSLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYANRTALFLDLL AQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMT LEPNKDLRPGDTVTITCSSYRGYPEAEVFWQDGQGAPLTGNVTTSQMA NEQGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQPMTF AAAHHHHHHHH 155Amino acid sequence of IGHV1-69*06 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWM GGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AR 156 Amino acid sequence of IGHJ6*01WGQGTTVTVSS 157 Amino acid sequence of IGKV1-9*01DIQLTQSPSFLSASVGDRVTITCR ASQGISSYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCQQLNSYPP 158Amino acid sequence of IGKJ2*01 FGQGTKLEIK 159Ig gamma-1 constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 160Ig gamma-1 constant region mutant ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 161Ig Kappa constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC 162 Ig Lambda constant regionQPKAAPSVTLFPPSSEELQANKAT LVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLS LTPEQWKSHRSYSCQVTHEGSTVE KTVAPTECS 163Amino acid sequence of IGKV6-21*01 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSLHWYQQKPDQSPKLLI KYASQSFSGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCHQSSSLPX 164 Amino acid sequence of IGKV2-28*01DIVMTQSPLSLPVTPGEPASISCR SSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGS GTDFTLKISRVEAEDVGVYYCMQA LQTPP 165Amino acid sequence of IGKJ4*01 FGGGTKVEIK 166Amino acid sequence of IGHV-b*01(0-1) QVQLQESGPGLVKPSETLSLTCAVSGYSISSGYYWGWIRQPPGKGLEW IGSIYHSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYC AR 167 Amino acid sequence of IGKv1-39*01DIQMTQSPSSLSASVGDRVTITCR ASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQSYSTPP 168Amino acid sequence of huAb13v1 heavy EVQLQESGPGLVKPSETLSLTCAV chainTGYSITSGYSWHWIRQFPGNGLEW Note: MGYIHSSGSTNYNPSLKSRISISRIg gamma-1 constant region mutant DTSKNQFFLKLSSVTAADTAVYYCsequence is underlined. AGYDDYFEYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVF LFPPKPKDTLMISRTPEVICVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK 169Amino acid sequence of huAb13v1 light DIQMTQSPSSLSASVGDRVTITCK chainASQNVGFNVAWYQQKPGKSPKALI Note: YSASYRYSGVPSRFSGSGSGTDFTIg kappa constant region sequence LTISSLQPEDFAEYFCQQYNWYPFis underlined. TFGQGTKLEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC170 Amino acid sequence of huAb3v2.5 heavy EVQLVQSGAEVKKPGSSVKVSCKAchain SGYTFSSYWMHWVRQAPGQGLEWI Note: GLIHPESGSTNYNEMFKNRATLTVIg gamma-1 constant region mutant DRSTSTAYMELSSLRSEDTAVYYCsequence is underlined. AGGGRLYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 171Amino acid sequence of huAb3v2.5 light DIVMTQSPLSLPVTPGEPASISCR chainSSQSLVHSNRDTYLRWYLQKPGQS Note: PQLLIYKVSNRFSGVPDRFSGSGSIg kappa constant region sequence GTDFTLKISRVEAEDVGVYYCSQSis underlined. THVPYTFGGGTKVEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC 172 Amino acid sequence of huAb3v2.6 heavyEVQLVQSGAEVKKPGSSVKVSCKA chain SGYTFSSYWMHWVRQAPGQGLEWI Note:GLIHPESGSTNYNEMFKNRATLTV Ig gamma-1 constant region mutant DRSTSTAYMsequence is underlined. ELSSLRSEDTAVYYCAGGGRLYFDYWGQGTTVTVSSASTKGPSVFPLA PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSL SLSPGK 173Amino acid sequence of huAb3v2.6 light DIVMTQSPLSLPVTPGEPASISCR chainSSQSLVHSNQDTYLRWYLQKPGQS Note: PQLLIYKVSNRFSGVPDRFSGSGSIg kappa constant region sequence GTDFTLKISRVEAEDVGVYYCSQSis underlined. THVPYTFGGGTKVEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC 174 Amino acid sequence ofQVQLVQSGAEVKKPGSSVKVSCKA IGHV1-69*06 IGHJ6 SGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITA DKSTSTAYMELSSLRSEDTAVYYC ARXXXXXXXXWGQGTTVTVSS175 Amino acid sequence of DIVMTQSPLSLPVTPGEPASISCR IGKV2-28*01 IGKJ4SSQSLLHSNGYNYLDWYLQKPGQS PQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCXXX XXXXXXFGGGTKVEIK 176Amino acid sequence of IGHV4-b IGHJ6 QVQLQESGPGLVKPSETLSLTCAVSGYSISSGYYWGWIRQPPGKGLEW IGSIYHSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYC ARXXXXXXXWGQGTTVTVSS 177Amino acid sequence of IGKV1-39 IGKJ2 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI YAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCXXXXXXXX XFGQGTKLEIK 178Amino acid sequence of huAb3 VL1 DIVMTQSPLSLPVTPGEPASISCR variantsSSQSLVHSXGDTYLRWYLQKPGQS Note: PQLLIYKVSNRFSGVPDRFSGSGSX can be any amino acid except: GTDFTLKISRVEAEDVGVYYCSQSM, C, N, D, or Q THVPYTFGGGTKVEIK 179 Amino acid sequence of huAb3 VL1DIVMTQSPLSLPVTPGEPASISCR variants SSQSLVHSNXDTYLRWYLQKPGQS Note:PQLLIYKVSNRFSGVPDRFSGSGS X can be any amino acid except:GTDFTLKISRVEAEDVGVYYCSQS M, C, G, S, N, or P THVPYTFGGGTKVEIK 180Amino acid sequence of huAb3 VH1b EVQLVQSGAEVKKPGSSVKVSCKA variantsSGYTFSSYWMHWVRQAPGQGLEWI Note: GLIHPXSGSTNYNEMFKNRATLTVX can be any amino acid except: DRSTSTAYMELSSLRSEDTAVYYCM, C, N, D, or Q AGGGRLYFDYWGQGTTVTVSS 181Amino acid sequence of huAb3 VH1b EVQLVQSGAEVKKPGSSVKVSCKA variantsSGYTFSSYWMHWVRQAPGQGLEWI Note: GLIHPDXGSTNYNEMFKNRATLTVX can be any amino acid except: DRSTSTAYMELSSLRSEDTAVYYCM, C, G, S, N, or P AGGGRLYFDYWGQGTTVTVSS 182chAb13 VL CDR3 amino acid sequence QQYNSYPFT

INCORPORATION BY REFERENCE

The contents of all references, patents, pending patent applications andpublished patents, cited throughout this application are herebyexpressly incorporated by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. An isolated anti-hB7H3 antibody, wherein the antibody comprises aheavy chain variable region comprising a CDR3 having an amino acidsequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO:35, and SEQ ID NO:
 27. and a light chain variable region comprising aCDR3 having an amino acid sequence selected from the group consisting ofof SEQ ID NO: 15, SEQ ID NO: 39, and SEQ ID NO: 31; a CDR2 having anamino acid sequence selected from the group consisting of SEQ ID NO: 7,SEQ ID NO: 38, and SEQ ID NO: 30; and a CDR1 having an amino acidsequence selected from the group consisting of SEQ ID NO: 136, SEQ IDNO: 138, SEQ ID NO: 37, SEQ ID NO: 14, and SEQ ID NO:
 29. 2-12.(canceled)
 13. The antibody according to claim 1, wherein the antibodyfurther comprises a human acceptor framework and said human acceptorframework comprises an amino acid sequence selected from the groupconsisting of SEQ ID 155, 156, 164, 165, 166, and
 167. 14-22. (canceled)23. The antibody according to claim 1, wherein the antibody furthercomprises a human acceptor framework and said human acceptor frameworkcomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 155 to
 158. 24-31. (canceled)
 32. The anti-hB7-H3 antibodyaccording to claim 1, wherein the antibody comprises a heavy chainvariable domain comprising an amino acid sequence set forth in SEQ IDNO: 139 and a light chain variable domain comprising an amino acidsequence set forth in SEQ ID NO:
 135. 33. (canceled)
 34. The anti-hB7-H3antibody according to claim 1, wherein the antibody comprises a heavychain variable domain comprising an amino acid sequence set forth in SEQID NO: 139 and a light chain variable domain comprising an amino acidsequence set forth in SEQ ID NO:
 137. 35. (canceled)
 36. The anti-hB7-H3antibody according to claim 1, wherein the antibody comprises a heavychain variable domain comprising an amino acid sequence set forth in SEQID NO: 147 and a light chain variable domain comprising an amino acidsequence set forth in SEQ ID NO:
 144. 37-129. (canceled)
 130. Theisolated antibody according to claim 1, wherein the antibody, or antigenbinding portion thereof, comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 168 and a light chain comprising the aminoacid sequence of SEQ ID NO:
 169. 131. The isolated antibody, accordingto claim 1, wherein the antibody comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 170 and a light chain comprising theamino acid sequence of SEQ ID NO:
 171. 132. The isolated antibodyaccording to claim 1, wherein the antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 172 and a light chaincomprising the amino acid sequence of SEQ ID NO:
 173. 133-137.(canceled)